RMH

Dimethylsulfoniopropionate (DMSP) is one of the Earth's most abundant organosulfur molecules, a signalling molecule, a key nutrient for marine microorganisms and the major precursor for gaseous dimethyl sulfide (DMS). DMS, another infochemical in signalling pathways, is important in global sulfur cycling and affects the Earth's albedo, and potentially climate, via sulfate aerosol and cloud condensation nuclei production. It was thought that only eukaryotes produce significant amounts of DMSP, but here we demonstrate that many marine heterotrophic bacteria also produce DMSP, probably using the same methionine (Met) transamination pathway as macroalgae and phytoplankton. We identify the first DMSP synthesis gene in any organism, dsyB, which encodes the key methyltransferase enzyme of this pathway and is a reliable reporter for bacterial DMSP synthesis in marine Alphaproteobacteria. DMSP production and dsyB transcription are upregulated by increased salinity, nitrogen limitation and lower temperatures in our model DMSP-producing bacterium Labrenzia aggregata LZB033. With significant numbers of dsyB homologues in marine metagenomes, we propose that bacteria probably make a significant contribution to oceanic DMSP production. Furthermore, because DMSP production is not solely associated with obligate phototrophs, the process need not be confined to the photic zones of marine environments and, as such, may have been underestimated.

We have developed a novel technique, named DamID, for the identification of DNA loci that interact in vivo with specific nuclear proteins in eukaryotes. By tethering Escherichia coli DNA adenine methyltransferase (Dam) to a chromatin protein, Dam can be targeted in vivo to native binding sites of this protein, resulting in local DNA methylation. Sites of methylation can subsequently be mapped using methylation-specific restriction enzymes or antibodies. We demonstrate the successful application of DamID both in Drosophila cell cultures and in whole flies. When Dam is tethered to the DNA-binding domain of GAL4, targeted methylation is limited to a region of a few kilobases surrounding a GAL4 binding sequence. Using DamID, we identified a number of expected and unexpected target loci for Drosophila heterochromatin protein 1. DamID has potential for genome-wide mapping of in vivo targets of chromatin proteins in various eukaryotes.

Since phages present a major challenge to survival in most environments, bacteria express a battery of anti-phage defences including CRISPR–Cas, restriction-modification and abortive infection systems. Such strategies are effective, but the phage genome—which encodes potentially inhibitory gene products—is still allowed to enter the cell. The safest way to preclude phage infection is to block initial phage adsorption to the cell. Here, we describe a cell-surface modification that blocks infection by certain phages. Strains of the opportunistic pathogen Pseudomonas aeruginosa express one of five different type IV pilins (T4P), two of which are glycosylated with O-antigen units or polymers of D-arabinofuranose. We propose that predation by bacteriophages that use T4P as receptors selects for strains that mask potential phage binding sites using glycosylation. Here, we show that both modifications protect P. aeruginosa from certain pilus-specific phages. Alterations to pilin sequence can also block phage infection, but glycosylation is considered less likely to create disadvantageous phenotypes. Through construction of chimeric phages, we show that specific phage tail proteins allow for infection of strains with glycosylated pili. These studies provide insight into first-line bacterial defences against predation and ways in which phages circumvent them, and provide a rationale for the prevalence of pilus glycosylation in nature.

Most studies of bacterial motility have examined small-scale (micrometer–centimeter) cell dispersal in monocultures. However, bacteria live in multispecies communities, where interactions with other microbes may inhibit or facilitate dispersal. Here, we demonstrate that motile bacteria in cheese rind microbiomes use physical networks created by filamentous fungi for dispersal, and that these interactions can shape microbial community structure. Serratia proteamaculans and other motile cheese rind bacteria disperse on fungal networks by swimming in the liquid layers formed on fungal hyphae. RNA-sequencing, transposon mutagenesis, and comparative genomics identify potential genetic mechanisms, including flagella-mediated motility, that control bacterial dispersal on hyphae. By manipulating fungal networks in experimental communities, we demonstrate that fungal-mediated bacterial dispersal can shift cheese rind microbiome composition by promoting the growth of motile over non-motile community members. Our single-cell to whole-community systems approach highlights the interactive dynamics of bacterial motility in multispecies microbiomes.

Secreted exopolysaccharides present important determinants for bacterial biofilm formation, survival, and virulence. Cellulose secretion typically requires the concerted action of a c-di-GMP-responsive inner membrane synthase (BcsA), an accessory membrane-anchored protein (BcsB), and several additional Bcs components. Although the BcsAB catalytic duo has been studied in great detail, its interplay with co-expressed subunits remains enigmatic. Here we show that E. coli Bcs proteins partake in a complex protein interaction network. Electron microscopy reveals a stable, megadalton-sized macromolecular assembly, which encompasses most of the inner membrane and cytosolic Bcs components and features a previously unobserved asymmetric architecture. Heterologous reconstitution and mutational analyses point toward a structure–function model, where accessory proteins regulate secretion by affecting both the assembly and stability of the system. Altogether, these results lay the foundation for more comprehensive models of synthase-dependent exopolysaccharide secretion in biofilms and add a sophisticated secretory nanomachine to the diverse bacterial arsenal for virulence and adaptation.

In this study we characterise three tandem promoters (PR1-1, PR1-2 and PR1-3) within the PR1 regulatory region of the Escherichia coli kps capsule gene cluster. Transcription from promoter PR1-2 was dependent on the activity of the upstream promoter PR1-1, which activated PR1-2 via transcription coupled DNA supercoiling. During growth at 37 °C a temporal pattern of transcription from all three promoters was observed with maximum transcriptional activity evident during mid-exponential phase followed by a sharp decrease in activity as the cells enter stationary phase. The growth phase dependent transcription was regulated by Integration Host Factor (IHF), which bound within the PR1 region to repress transcription from PR1-2 and PR1-3. This pattern of transcription was mirrored by growth phase dependent expression of the K1 capsule. Overall these data reveal a complex pattern of transcriptional regulation for an important virulence factor with IHF playing a role in regulating growth phase expression.

Sensitive detection of off-target effects is important for translating CRISPR–Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR–Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequences. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR–Cas9.

The cell envelope of gram-negative bacteria, a structure comprising an outer (OM) and an inner (IM) membrane, is essential for life. The OM and the IM are separated by the periplasm, a compartment that contains the peptidoglycan. The OM is tethered to the peptidoglycan via the lipoprotein, Lpp. However, the importance of the envelope’s multilayered architecture remains unknown. Here, when we removed physical coupling between the OM and the peptidoglycan, cells lost the ability to sense defects in envelope integrity. Further experiments revealed that the critical parameter for the transmission of stress signals from the envelope to the cytoplasm, where cellular behaviour is controlled, is the IM-to-OM distance. Augmenting this distance by increasing the length of the lipoprotein Lpp destroyed signalling, whereas simultaneously increasing the length of the stress-sensing lipoprotein RcsF restored signalling. Our results demonstrate the physiological importance of the size of the periplasm. They also reveal that strict control over the IM-to-OM distance is required for effective envelope surveillance and protection, suggesting that cellular architecture and the structure of transenvelope protein complexes have been evolutionarily co-optimised for correct function. Similar strategies are likely at play in cellular compartments surrounded by 2 concentric membranes, such as chloroplasts and mitochondria.

Plant RuBisCo, a complex of eight large and eight small subunits, catalyzes the fixation of CO2 in photosynthesis. The low catalytic efficiency of RuBisCo provides strong motivation to reengineer the enzyme with the goal of increasing crop yields. However, genetic manipulation has been hampered by the failure to express plant RuBisCo in a bacterial host. We achieved the functional expression of Arabidopsis thaliana RuBisCo in Escherichia coli by coexpressing multiple chloroplast chaperones. These include the chaperonins Cpn60/Cpn20, RuBisCo accumulation factors 1 and 2, RbcX, and bundle-sheath defective-2 (BSD2). Our structural and functional analysis revealed the role of BSD2 in stabilizing an end-state assembly intermediate of eight RuBisCo large subunits until the small subunits become available. The ability to produce plant RuBisCo recombinantly will facilitate efforts to improve the enzyme through mutagenesis.

Rhizobium sp. IRBG74 not only nodulates Sesbania cannabina but also can enhance rice growth; however, the underlying molecular mechanisms are not clear. Here, we show that Rhizobium sp. IRBG74 colonizes the roots of Arabidopsis thaliana, which leads to inhibition in the growth of main root but enhancement in the formation of lateral roots. The promotion of lateral root formation by Rhizobium sp. IRBG74 in the fls2-1 mutant, which is insensitive to flagellin, is similar to the wild-type plant, while the auxin response deficient mutant tir1-1 is significantly less sensitive to Rhizobium sp. IRBG74 than the wild type in terms of the inhibition of main root elongation and the promotion of lateral root formation. Further transcriptome analysis of Arabidopsis roots inoculated with Rhizobium sp. IRBG74 revealed differential expression of 50 and 211 genes at 24 and 48 h, respectively, and a majority of these genes are involved in auxin signaling. Consistent with the transcriptome analysis results, Rhizobium sp. IRBG74 treatment induces expression of the auxin responsive reporter DR5:GUS in roots. Our results suggest that in Arabidopsis Rhizobium sp. IRBG74 colonizes roots and promotes the lateral root formation likely through modulating auxin signaling. Our work provides insight into the molecular mechanisms of interactions between legume-nodulating rhizobia and non-legume plants.

The occurrence of mutations is a cornerstone of the evolutionary theory of adaptation, capitalizing on the rare chance that a mutation confers a fitness benefit. Natural selection is increasingly being leveraged in laboratory settings for industrial and basic science applications. Despite increasing deployment, there are no standardized procedures available for designing and performing adaptive laboratory evolution (ALE) experiments. Thus, there is a need to optimize the experimental design, specifically for determining when to consider an experiment complete and for balancing outcomes with available resources (i.e., laboratory supplies, personnel, and time). To design and to better understand ALE experiments, a simulator, ALEsim, was developed, validated, and applied to the optimization of ALE experiments. The effects of various passage sizes were experimentally determined and subsequently evaluated with ALEsim, to explain differences in experimental outcomes. Furthermore, a beneficial mutation rate of 10−6.9 to 10−8.4 mutations per cell division was derived. A retrospective analysis of ALE experiments revealed that passage sizes typically employed in serial passage batch culture ALE experiments led to inefficient production and fixation of beneficial mutations. ALEsim and the results described here will aid in the design of ALE experiments to fit the exact needs of a project while taking into account the resources required and will lower the barriers to entry for this experimental technique.

Quorum sensing (QS) is a cell–cell communication process that enables bacteria to track cell population density and orchestrate collective behaviors. QS relies on the production and detection of, and the response to, extracellular signal molecules called autoinducers. In Vibrio cholerae, multiple QS circuits control pathogenesis and biofilm formation. Here, we identify and characterize a new QS autoinducer–receptor pair. The autoinducer is 3,5-dimethylpyrazin-2-ol (DPO). DPO is made from threonine and alanine, and its synthesis depends on threonine dehydrogenase (Tdh). DPO binds to and activates a transcription factor, VqmA. The VqmA–DPO complex activates expression of vqmR, which encodes a small regulatory RNA. VqmR represses genes required for biofilm formation and toxin production. We propose that DPO allows V. cholerae to regulate collective behaviors to, among other possible roles, diversify its QS output during colonization of the human host.