Plasmids are mobile genetic elements that play a key role in microbial ecology and evolution by mediating horizontal transfer of important genes, such as antimicrobial resistance genes. Many microbial genomes have been sequenced by short read sequencers and have resulted in a mix of contigs that derive from plasmids or chromosomes. New tools that accurately identify plasmids are needed to elucidate new plasmid-borne genes of high biological importance. We have developed Deeplasmid, a deep learning tool for distinguishing plasmids from bacterial chromosomes based on the DNA sequence and its encoded biological data. It requires as input only assembled sequences generated by any sequencing platform and assembly algorithm and its runtime scales linearly with the number of assembled sequences. Deeplasmid achieves an AUC–ROC of over 89%, and it was more accurate than five other plasmid classification methods. Finally, as a proof of concept, we used Deeplasmid to predict new plasmids in the fish pathogen Yersinia ruckeri ATCC 29473 that has no annotated plasmids. Deeplasmid predicted with high reliability that a long assembled contig is part of a plasmid. Using long read sequencing we indeed validated the existence of a 102 kb long plasmid, demonstrating Deeplasmid's ability to detect novel plasmids.

The extracellular Contractile Injection System (eCIS) is a toxin-delivery particle that evolved from a bacteriophage tail. Four eCISs have previously been shown to mediate interactions between bacteria and their invertebrate hosts. Here, we identify eCIS loci in 1,249 bacterial and archaeal genomes and reveal an enrichment of these loci in environmental microbes and their apparent absence from mammalian pathogens. We show that 13 eCIS-associated toxin genes from diverse microbes can inhibit the growth of bacteria and/or yeast. We identify immunity genes that protect bacteria from self-intoxication, further supporting an antibacterial role for some eCISs. We also identify previously undescribed eCIS core genes, including a conserved eCIS transcriptional regulator. Finally, we present our data through an extensive eCIS repository, termed eCIStem. Our findings support eCIS as a toxin-delivery system that is widespread among environmental prokaryotes and likely mediates antagonistic interactions with eukaryotes and other prokaryotes.

Fruit flies in the genus Bactrocera are among the most destructive insect pests of fruits and vegetables throughout the world. A number of studies have identified volatiles from fruit flies, but few reports have demonstrated behavioral effects or sensitivities of fly antennae to these compounds. We applied a recently developed method of automated headspace analysis using SPME (Solid Phase Microextraction) fibers and GC MS (gas chromatography mass spectrometry), termed SSGA, to reveal volatiles specific to each sex of B. zonata that are emitted in a diel periodicity. The volatiles released primarily at dusk were identified by GC MS and chemical syntheses as several spiroacetals, pyrazines, and ethyl esters. Solvent extraction of male rectal glands or airborne collections from each sex, followed by GC MS, showed that certain of the volatiles increase or decrease in quantity sex-specifically with age of the flies. Electroantennographic (EAG) analysis of dose-response indicates differences in sensitivities of male and female antenna to the various volatiles. Our study provides a comprehensive analysis of the volatile chemicals produced and released by B. zonata and their antennal responses. The possible pheromone and semiochemical roles of the various volatiles released by each sex and the difficulties of establishing behavioral functions are discussed.

Endophytes are microorganisms that live inside plants and are often beneficial for the host. Kosakonia is a novel bacterial genus that includes several species that are diazotrophic and plant associated. This study revealed two quorum sensing-related LuxR solos, designated LoxR and PsrR, in the plant endophyte Kosakonia sp. strain KO348. LoxR modeling and biochemical studies demonstrated that LoxR binds N-acyl homoserine lactones (AHLs) in a promiscuous way. PsrR, on the other hand, belongs to the subfamily of plant-associated-bacterium (PAB) LuxR solos that respond to plant compounds. Target promoter studies as well as modeling and phylogenetic comparisons suggest that PAB LuxR solos are likely to respond to different plant compounds. Finally, LoxR is involved in the regulation of T6SS and PsrR plays a role in root endosphere colonization. IMPORTANCE Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling.

Beneficial rhizobacteria dwell in plant roots and promote plant growth, development, and resistance to various stress types. In recent years there have been large-scale efforts to culture root-associated bacteria and sequence their genomes to uncover novel beneficial microbes. However, only a few strains of rhizobacteria from the large pool of soil microbes have been studied at the molecular level. This review focuses on the molecular basis underlying the phenotypes of three beneficial microbe groups; (1) plant-growth promoting rhizobacteria (PGPR), (2) root nodulating bacteria (RNB), and (3) biocontrol agents (BCAs). We focus on bacterial proteins and secondary metabolites that mediate known phenotypes within and around plants, and the mechanisms used to secrete these. We highlight the necessity for a better understanding of bacterial genes responsible for beneficial plant traits, which can be used for targeted gene-centered and molecule-centered discovery and deployment of novel beneficial rhizobacteria.

Azelaic acid is a dicarboxylic acid that has recently been shown to play a role in plant-bacteria signalling and also occurs naturally in several cereals. Several bacteria have been reported to be able to utilize azelaic acid as a unique source of carbon and energy, including Pseudomonas nitroreducens . In this study, we utilize P. nitroreducens as a model organism to study bacterial degradation of and response to azelaic acid. We report genetic evidence of azelaic acid degradation and the identification of a transcriptional regulator that responds to azelaic acid in P. nitroreducens DSM 9128. Three mutants possessing transposons in genes of an acyl-CoA ligase, an acyl-CoA dehydrogenase and an isocitrate lyase display a deficient ability in growing in azelaic acid. Studies on transcriptional regulation of these genes resulted in the identification of an IclR family repressor that we designated as AzeR, which specifically responds to azelaic acid. A bioinformatics survey reveals that AzeR is confined to a few proteobacterial genera that are likely to be able to degrade and utilize azelaic acid as the sole source of carbon and energy.

Endophytes live inside plants and are often beneficial. Kosakonia is a novel bacterial genus that includes many diazotrophic plant-associated isolates. Plant–bacteria studies on two rice endophytic Kosakonia beneficial strains were performed, including comparative genomics, secretome profiling, in planta tests, and a field release trial. The strains are efficient rhizoplane and root endosphere colonizers and localized in the root cortex. Secretomics revealed 144 putative secreted proteins, including type VI secretory system (T6SS) proteins. A Kosakonia T6SS genomic knock-out mutant showed a significant decrease in rhizoplane and endosphere colonization ability. A field trial using rice seed inoculated with Kosakonia spp. showed no effect on plant growth promotion upon nitrogen stress and microbiome studies revealed that Kosakonia spp. were significantly more present in the inoculated rice. Comparative genomics indicated that several protein domains were enriched in plant-associated Kosakonia spp. This study highlights that Kosakonia is an important, recently classified genus involved in plant–bacteria interaction.

There is a growing appreciation for the important roles microorganisms play in association with plants. Microorganisms are drawn to distinct plant surfaces by the nutrient-rich microenvironment, and in turn some of these colonizing microbes provide mutualistic benefits to their host. The development of plant probiotics to increase crop yield and provide plant resistance against biotic and abiotic stresses, while minimizing chemical inputs, would benefit from a deeper mechanistic understanding of plant-microbe interaction. Technological advances in molecular biology and high-throughput -omics provide stepping stones to the elucidation of critical microbiome gene functions that aid in improving plant performance. Here, we review -omics-based approaches that are propelling forward the current understanding of plant-associated bacterial gene functions, and describe how these technologies have helped unravel key bacterial genes and pathways that mediate pathogenic, beneficial, and commensal host interactions.

Plants intimately associate with diverse bacteria. Plant-associatedbacteria have ostensibly evolved genes that enable them to adapt to plantenvironments. However, the identities of such genes are mostly unknown, and theirfunctions are poorly characterized. We sequenced 484 genomes of bacterial isolatesfrom roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterialgenomes to identify thousands of plant-associated gene clusters. Genomes ofplant-associated bacteria encode more carbohydrate metabolism functions and fewermobile elements than related non-plant-associated genomes do. We experimentallyvalidated candidates from two sets of plant-associated genes: one involved in plantcolonization, and the other serving in microbe–microbe competition betweenplant-associated bacteria. We also identified 64 plant-associated protein domainsthat potentially mimic plant domains; some are shared with plant-associated fungiand oomycetes. This work expands the genome-based understanding of plant–microbeinteractions and provides potential leads for efficient and sustainable agriculturethrough microbiome engineering.

The emergence of mutations following growth-limiting conditions underlies bacterial drug resistance, viral escape from the immune system and fundamental evolution-driven events. Intriguingly, whether mutations are induced by growth limitation conditions or are randomly generated during growth and then selected by growth limitation conditions remains an open question1. Here, we show that bacteriophage T7 undergoes apparent stress-induced mutagenesis when selected for improved recognition of its host's receptor. In our unique experimental set-up, the growth limitation condition is physically and temporally separated from mutagenesis: growth limitation occurs while phage DNA is outside the host, and spontaneous mutations occur during phage DNA replication inside the host. We show that the selected beneficial mutations are not pre-existing and that the initial slow phage growth is enabled by the phage particle's low-efficiency DNA injection into the host. Thus, the phage particle allows phage populations to initially extend their host range without mutagenesis by virtue of residual recognition of the host receptor. Mutations appear during non-selective intracellular replication, and the frequency of mutant phages increases by natural selection acting on free phages, which are not capable of mutagenesis.

Over the past decade, high-throughput short-read 16S rRNA gene amplicon sequencing has eclipsed clone-dependent long-read Sanger sequencing for microbial community profiling. The transition to new technologies has provided more quantitative information at the expense of taxonomic resolution with implications for inferring metabolic traits in various ecosystems. We applied single-molecule real-time sequencing for microbial community profiling, generating full-length 16S rRNA gene sequences at high throughput, which we propose to name PhyloTags. We benchmarked and validated this approach using a defined microbial community. When further applied to samples from the water column of meromictic Sakinaw Lake, we show that while community structures at the phylum level are comparable between PhyloTags and Illumina V4 16S rRNA gene sequences (iTags), variance increases with community complexity at greater water depths. PhyloTags moreover allowed less ambiguous classification. Last, a platform-independent comparison of PhyloTags and in silico generated partial 16S rRNA gene sequences demonstrated significant differences in community structure and phylogenetic resolution across multiple taxonomic levels, including a severe underestimation in the abundance of specific microbial genera involved in nitrogen and methane cycling across the Lake’s water column. Thus, PhyloTags provide a reliable adjunct or alternative to cost-effective iTags, enabling more accurate phylogenetic resolution of microbial communities and predictions on their metabolic potential.

Summary Toxin-antitoxin modules are gene pairs encoding a toxin and its antitoxin, and are found on the chromosomes of many bacteria, including pathogens. Here, we characterize the specific contribution of the TxpA and YqcG toxins in elimination of defective cells from developing Bacillus subtilis biofilms. On nutrient limitation, defective cells accumulated in the biofilm breaking its symmetry. Deletion of the toxins resulted in accumulation of morphologically abnormal cells, and interfered with the proper development of the multicellular community. Dual physiological responses are of significance for TxpA and YqcG activation: nitrogen deprivation enhances the transcription of both TxpA and YqcG toxins, and simultaneously sensitizes the biofilm cells to their activity. Furthermore, we demonstrate that while both toxins when overexpressed affect the morphology of the developing biofilm, the toxin TxpA can act to lyse and dissolve pre-established B. subtilis biofilms.