Genome-wide gene expression changes of Pseudomonas veronii 1YdBTEX2 during bioaugmentation in polluted soils.

Bioaugmentation aims to use the capacities of specific bacterial strains inoculated into sites to enhance pollutant biodegradation. Bioaugmentation results have been mixed, which has been attributed to poor inoculant growth and survival in the field, and, consequently, moderate catalytic performance. However, our understanding of biodegradation activity mostly comes from experiments conducted under laboratory conditions, and the processes occurring during adaptation and invasion of inoculants into complex environmental microbiomes remain poorly known. The main aim of this work was thus to study the specific and different cellular reactions of an inoculant for bioaugmentation during adaptation, growth and survival in natural clean and contaminated non-sterile soils, in order to better understand factors limiting bioaugmentation. As inoculant we focused on the monoaromatic compound-degrading bacterium Pseudomonas veronii 1YdBTEX2. The strain proliferated in all but one soil types in presence and in absence of exogenously added toluene. RNAseq and differential genome-wide gene expression analysis illustrated both a range of common soil responses such as increased nutrient scavenging and recycling, expression of defense mechanisms, as well as environment-specific reactions, notably osmoprotection and metal homeostasis. The core metabolism of P. veronii remained remarkably constant during exponential growth irrespective of the environment, with slight changes in cofactor regeneration pathways, possibly needed for balancing defense reactions. P. veronii displayed a versatile global program, enabling it to adapt to a variety of soil environments in the presence and even in absence of its target pollutant toluene. Our results thus challenge the widely perceived dogma of poor survival and growth of exogenous inoculants in complex microbial ecosystems such as soil and provide a further basis to developing successful bioaugmentation strategies

Draft Genome Sequence of Plantibacterflavus Strain 251 Isolated from a Plant Growing in a Chronically Hydrocarbon-Contaminated Site.

Plantibacter flavus isolate 251 is a bacterial endophyte isolated from an Achillea millefolium plant growing in a natural oil seep soil located in Oil Springs, Ontario, Canada. We present here a draft genome sequence of an infrequently reported genus Plantibacter, highlighting an endophytic lifestyle and biotechnological potential

Physiological and transcriptome changes induced by Pseudomonas putida acquisition of an integrative and conjugative element.

Integrative and conjugative elements (ICEs) comprise ubiquitous large mobile regions in prokaryotic chromosomes that transmit vertically to daughter cells and transfer horizontally to distantly related lineages. Their evolutionary success originates in maximized combined ICE-host fitness trade-offs, but how the ICE impacts on the host metabolism and physiology is poorly understood. Here we investigate global changes in the host genetic network and physiology of Pseudomonas putida with or without an integrated ICEclc, a model ICE widely distributed in proteobacterial genomes. Genome-wide gene expression differences were analyzed by RNA-seq using exponentially growing or stationary phase-restimulated cultures on 3-chlorobenzoate, an aromatic compound metabolizable thanks to specific ICEclc-located genes. We found that the presence of ICEclc imposes a variety of changes in global pathways such as cell cycle and amino acid metabolism, which were more numerous in stationary-restimulated than exponential phase cells. Unexpectedly, ICEclc stimulates cellular motility and leads to more rapid growth on 3-chlorobenzoate than cells carrying only the integrated clc genes. ICEclc also concomitantly activates the P. putida Pspu28-prophage, but this in itself did not provoke measurable fitness effects. ICEclc thus interferes in a number of cellular pathways, inducing both direct benefits as well as indirect costs in P. putida

Draft Genome Sequence of Microbacterium foliorum Strain 122 Isolated from a Plant Growing in a Chronically Hydrocarbon-Contaminated Site.

Microbacterium foliorum strain 122 is a bacterial endophyte isolated from a Dactylis glomerata plant growing in a natural oil seep soil located in Oil Springs, Ontario, Canada. We present here a draft genome sequence of an endophytic strain that has promising potential in hydrocarbon degradation and plant growth promotion

Insights into Mobile Genetic Elements of the Biocide-Degrading Bacterium Pseudomonas nitroreducens HBP-1.

The sewage sludge isolate Pseudomonas nitroreducens HBP-1 was the first bacterium known to completely degrade the fungicide 2-hydroxybiphenyl. PacBio and Illumina whole-genome sequencing revealed three circular DNA replicons: a chromosome and two plasmids. Plasmids were shown to code for putative adaptive functions such as heavy metal resistance, but with unclarified ability for self-transfer. About one-tenth of strain HBP-1's chromosomal genes are likely of recent horizontal influx, being part of genomic islands, prophages and integrative and conjugative elements (ICEs). P. nitroreducens carries two large ICEs with different functional specialization, but with homologous core structures to the well-known ICEclc of Pseudomonas knackmussii B13. The variable regions of ICEPni1 (96 kb) code for, among others, heavy metal resistances and formaldehyde detoxification, whereas those of ICEPni2 (171 kb) encodes complete meta-cleavage pathways for catabolism of 2-hydroxybiphenyl and salicylate, a protocatechuate pathway and peripheral enzymes for 4-hydroxybenzoate, ferulate, vanillin and vanillate transformation. Both ICEs transferred at frequencies of 10 <sup>-6</sup> -10 <sup>-8</sup> per P. nitroreducens HBP-1 donor into Pseudomonas putida, where they integrated site specifically into tRNA <sup>Gly</sup> -gene targets, as expected. Our study highlights the underlying determinants and mechanisms driving dissemination of adaptive properties allowing bacterial strains to cope with polluted environments

Reproducible Propagation of Species-Rich Soil Bacterial Communities Suggests Robust Underlying Deterministic Principles of Community Formation

Microbiomes are typically characterized by high species diversity but it is poorly understood how such system-level complexity can be generated and propagated. Here, we used soil microcosms as a model to study development of bacterial communities as a function of their starting complexity and environmental boundary conditions. Despite inherent stochastic variation in manipulating species-rich communities, both laboratory-mixed medium complexity (21 soil bacterial isolates in equal proportions) and high-diversity natural top-soil communities followed highly reproducible succession paths, maintaining 16S rRNA gene amplicon signatures prominent for known soil communities in general. Development trajectories and compositional states were different for communities propagated in soil microcosms than in liquid suspension. Compositional states were maintained over multiple renewed growth cycles but could be diverged by short-term pollutant exposure. The different but robust trajectories demonstrated that deterministic taxa-inherent characteristics underlie reproducible development and self-organized complexity of soil microbiomes within their environmental boundary conditions. Our findings also have direct implications for potential strategies to achieve controlled restoration of desertified land. IMPORTANCE There is now a great awareness of the high diversity of most environmental ("free-living") and host-associated microbiomes, but exactly how diverse microbial communities form and maintain is still highly debated. A variety of theories have been put forward, but testing them has been problematic because most studies have been based on synthetic communities that fail to accurately mimic the natural composition (i.e., the species used are typically not found together in the same environment), the diversity (usually too low to be representative), or the environmental system itself (using designs with single carbon sources or solely mixed liquid cultures). In this study, we show how species-diverse soil bacterial communities can reproducibly be generated, propagated, and maintained, either from individual isolates (21 soil bacterial strains) or from natural microbial mixtures washed from top-soil. The high replicate consistency we achieve both in terms of species compositions and developmental trajectories demonstrates the strong inherent deterministic factors driving community formation from their species composition. Generating complex soil microbiomes may provide ways for restoration of damaged soils that are prevalent on our planet

Transcriptome analysis of the mobile genome ICEclc in Pseudomonas knackmussii B13

<p>Abstract</p> <p>Background</p> <p>Integrative and conjugative elements (ICE) form a diverse group of DNA elements that are integrated in the chromosome of the bacterial host, but can occasionally excise and horizontally transfer to a new host cell. ICE come in different families, typically with a conserved core for functions controlling the element's behavior and a variable region providing auxiliary functions to the host. The ICE<it>clc </it>element of <it>Pseudomonas knackmussii </it>strain B13 is representative for a large family of chromosomal islands detected by genome sequencing approaches. It provides the host with the capacity to degrade chloroaromatics and 2-aminophenol.</p> <p>Results</p> <p>Here we study the transcriptional organization of the ICE<it>clc </it>core region. By northern hybridizations, reverse-transcriptase polymerase chain reaction (RT-PCR) and Rapid Amplification of cDNA Ends (5'-RACE) fifteen transcripts were mapped in the core region. The occurrence and location of those transcripts were further confirmed by hybridizing labeled cDNA to a semi-tiling micro-array probing both strands of the ICE<it>clc </it>core region. Dot blot and semi-tiling array hybridizations demonstrated most of the core transcripts to be upregulated during stationary phase on 3-chlorobenzoate, but not on succinate or glucose.</p> <p>Conclusions</p> <p>The transcription analysis of the ICE<it>clc </it>core region provides detailed insights in the mode of regulatory organization and will help to further understand the complex mode of behavior of this class of mobile elements. We conclude that ICE<it>clc </it>core transcription is concerted at a global level, more reminiscent of a phage program than of plasmid conjugation.</p

Complete alanine scanning of the Escherichia coli RbsB ribose binding protein reveals residues important for chemoreceptor signaling and periplasmic abundance.

The Escherichia coli RbsB ribose binding protein has been used as a scaffold for predicting new ligand binding functions through in silico modeling, yet with limited success and reproducibility. In order to possibly improve the success of predictive modeling on RbsB, we study here the influence of individual residues on RbsB-mediated signaling in a near complete library of alanine-substituted RbsB mutants. Among a total of 232 tested mutants, we found 10 which no longer activated GFPmut2 reporter expression in E. coli from a ribose-RbsB hybrid receptor signaling chain, and 13 with significantly lower GFPmut2 induction than wild-type. Quantitative mass spectrometry abundance measurements of 25 mutants and wild-type RbsB in periplasmic space showed four categories of effects. Some (such as D89A) seem correctly produced and translocated but fail to be induced with ribose. Others (such as N190A) show lower induction probably as a result of less efficient production, folding and translocation. The third (such as N41A or K29A) have defects in both induction and abundance. The fourth category consists of semi-constitutive mutants with increased periplasmic abundance but maintenance of ribose induction. Our data show how RbsB modeling should include ligand-binding as well as folding, translocation and receptor binding

Mechanistic insights into bacterial metabolic reprogramming from omics-integrated genome-scale models.

Understanding the adaptive responses of individual bacterial strains is crucial for microbiome engineering approaches that introduce new functionalities into complex microbiomes, such as xenobiotic compound metabolism for soil bioremediation. Adaptation requires metabolic reprogramming of the cell, which can be captured by multi-omics, but this data remains formidably challenging to interpret and predict. Here we present a new approach that combines genome-scale metabolic modeling with transcriptomics and exometabolomics, both of which are common tools for studying dynamic population behavior. As a realistic demonstration, we developed a genome-scale model of Pseudomonas veronii 1YdBTEX2, a candidate bioaugmentation agent for accelerated metabolism of mono-aromatic compounds in soil microbiomes, while simultaneously collecting experimental data of P. veronii metabolism during growth phase transitions. Predictions of the P. veronii growth rates and specific metabolic processes from the integrated model closely matched experimental observations. We conclude that integrative and network-based analysis can help build predictive models that accurately capture bacterial adaptation responses. Further development and testing of such models may considerably improve the successful establishment of bacterial inoculants in more complex systems

ICEApl1, an integrative conjugative element related to ICEHin1056, identified in the pig pathogen Actinobacillus pleuropneumoniae

ICEApl1 was identified in the whole genome sequence of MIDG2331, a tetracycline-resistant (MIC = 8 mg/L) serovar 8 clinical isolate of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. PCR amplification of virB4, one of the core genes involved in conjugation, was used to identify other A. pleuropneumoniae isolates potentially carrying ICEApl1. MICs for tetracycline were determined for virB4 positive isolates, and shotgun whole genome sequence analysis was used to confirm presence of the complete ICEApl1. The sequence of ICEApl1 is 56083 bp long and contains 67 genes including a Tn10 element encoding tetracycline resistance. Comparative sequence analysis was performed with similar integrative conjugative elements (ICEs) found in other members of the Pasteurellaceae. ICEApl1 is most similar to the 59393 bp ICEHin1056, from Haemophilus influenzae strain 1056. Although initially identified only in serovar 8 isolates of A. pleuropneumoniae (31 from the UK and 1 from Cyprus), conjugal transfer of ICEApl1 to representative isolates of other serovars was confirmed. All isolates carrying ICEApl1 had a MIC for tetracycline of 8 mg/L. This is, to our knowledge, the first description of an ICE in A. pleuropneumoniae, and the first report of a member of the ICEHin1056 subfamily in a non-human pathogen. ICEApl1 confers resistance to tetracycline, currently one of the more commonly used antibiotics for treatment and control of porcine pleuropneumonia