Pseudomonas putida mediates bacterial killing, biofilm invasion and biocontrol with a type IVB secretion system.

Many bacteria utilize contact-dependent killing machineries to eliminate rivals in their environmental niches. Here we show that the plant root colonizer Pseudomonas putida strain IsoF is able to kill a wide range of soil and plant-associated Gram-negative bacteria with the aid of a type IVB secretion system (T4BSS) that delivers a toxic effector into bacterial competitors in a contact-dependent manner. This extends the range of targets of T4BSSs-so far thought to transfer effectors only into eukaryotic cells-to prokaryotes. Bioinformatic and genetic analyses showed that this killing machine is entirely encoded by the kib gene cluster located within a rare genomic island, which was recently acquired by horizontal gene transfer. P. putida IsoF utilizes this secretion system not only as a defensive weapon to kill bacterial competitors but also as an offensive weapon to invade existing biofilms, allowing the strain to persist in its natural environment. Furthermore, we show that strain IsoF can protect tomato plants against the phytopathogen Ralstonia solanacearum in a T4BSS-dependent manner, suggesting that IsoF can be exploited for pest control and sustainable agriculture.

Paraburkholderia phymatum Homocitrate Synthase NifV Plays a Key Role for Nitrogenase Activity during Symbiosis with Papilionoids and in Free-Living Growth Conditions.

Homocitrate is an essential component of the iron-molybdenum cofactor of nitrogenase, the bacterial enzyme that catalyzes the reduction of dinitrogen (N2) to ammonia. In nitrogen-fixing and nodulating alpha-rhizobia, homocitrate is usually provided to bacteroids in root nodules by their plant host. In contrast, non-nodulating free-living diazotrophs encode the homocitrate synthase (NifV) and reduce N2 in nitrogen-limiting free-living conditions. Paraburkholderia phymatum STM815 is a beta-rhizobial strain, which can enter symbiosis with a broad range of legumes, including papilionoids and mimosoids. In contrast to most alpha-rhizobia, which lack nifV, P. phymatum harbors a copy of nifV on its symbiotic plasmid. We show here that P. phymatum nifV is essential for nitrogenase activity both in root nodules of papilionoid plants and in free-living growth conditions. Notably, nifV was dispensable in nodules of Mimosa pudica despite the fact that the gene was highly expressed during symbiosis with all tested papilionoid and mimosoid plants. A metabolome analysis of papilionoid and mimosoid root nodules infected with the P. phymatum wild-type strain revealed that among the approximately 400 measured metabolites, homocitrate and other metabolites involved in lysine biosynthesis and degradation have accumulated in all plant nodules compared to uninfected roots, suggesting an important role of these metabolites during symbiosis.

Investigation of Burkholderia cepacia Complex Methylomes via Single-Molecule, Real-Time Sequencing and Mutant Analysis.

Bacterial genomes can be methylated at particular motifs by methyltransferases (MTs). This DNA modification allows restriction endonucleases (REs) to discriminate between self and foreign DNA. While the accepted primary function of such restriction modification (RM) systems is to degrade incoming foreign DNA, other roles of RM systems and lone RE or MT components have been found in genome protection, stability, and the regulation of various phenotypes. The Burkholderia cepacia complex (Bcc) is a group of closely related opportunistic pathogens with biotechnological potential. Here, we constructed and analyzed mutants lacking various RM components in the clinical Bcc isolate Burkholderia cenocepacia H111 and used single-molecule, real-time (SMRT) sequencing of single mutants to assign the B. cenocepacia H111 MTs to their cognate motifs. DNA methylation is shown to affect biofilm formation, cell shape, motility, siderophore production, and membrane vesicle production. Moreover, DNA methylation had a large effect on the maintenance of the Bcc virulence megaplasmid pC3. Our data also suggest that the gp51 MT-encoding gene, which is essential in H111 and is located within a prophage, is required for maintaining the bacteriophage in a lysogenic state, thereby ensuring a constant, low level of phage production within the bacterial population. IMPORTANCE While the genome sequence determines an organism's proteins, methylation of the nucleotides themselves can confer additional properties. In bacteria, MTs modify specific nucleotide motifs to allow discrimination of "self" from "nonself" DNA, e.g., from bacteriophages. Restriction enzymes detect "nonself" methylation patterns and cut foreign DNA. Furthermore, methylation of promoter regions can influence gene expression and hence affect various phenotypes. In this study, we determined the methylated motifs of four strains from the Burkholderia cepacia complex of opportunistic pathogens. We deleted all genes encoding the restriction and modification components in one of these strains, Burkholderia cenocepacia H111. It is shown that DNA methylation affects various phenotypic traits, the most noteworthy being lysogenicity of a bacteriophage and maintenance of a virulence megaplasmid.

Detection of cytosine methylation in Burkholderia cenocepacia by single-molecule real-time sequencing and whole-genome bisulfite sequencing.

Research on prokaryotic epigenetics, the study of heritable changes in gene expression independent of sequence changes, led to the identification of DNA methylation as a versatile regulator of diverse cellular processes. Methylation of adenine bases is often linked to regulation of gene expression in bacteria, but cytosine methylation is also frequently observed. In this study, we present a complete overview of the cytosine methylome in Burkholderia cenocepacia, an opportunistic respiratory pathogen in cystic fibrosis patients. Single-molecule real-time (SMRT) sequencing was used to map all 4mC-modified cytosines, as analysis of the predicted MTases in the B. cenocepacia genome revealed the presence of a 4mC-specific phage MTase, M.BceJII, targeting GGCC sequences. Methylation motif GCGGCCGC was identified, and out of 6850 motifs detected across the genome, 2051 (29.9 %) were methylated at the fifth position. Whole-genome bisulfite sequencing (WGBS) was performed to map 5mC methylation and 1635 5mC-modified cytosines were identified in CpG motifs. A comparison of the genomic positions of the modified bases called by each method revealed no overlap, which confirmed the authenticity of the detected 4mC and 5mC methylation by SMRT sequencing and WGBS, respectively. Large inter-strain variation of the 4mC-methylated cytosines was observed when B. cenocepacia strains J2315 and K56-2 were compared, which suggests that GGCC methylation patterns in B. cenocepacia are strain-specific. It seems likely that 4mC methylation of GGCC is not involved in regulation of gene expression but rather is a remnant of bacteriophage invasion, in which methylation of the phage genome was crucial for protection against restriction-modification systems of B. cenocepacia.

DNA Methylation Epigenetically Regulates Gene Expression in Burkholderia cenocepacia and Controls Biofilm Formation, Cell Aggregation, and Motility.

Respiratory tract infections by the opportunistic pathogen Burkholderia cenocepacia often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to tackle these infections might emerge from the field of epigenetics, as DNA methylation is an important regulator of gene expression. The present study focused on two DNA methyltransferases (MTases) in B. cenocepacia strains J2315 and K56-2 and their role in regulating gene expression. In silico predicted DNA MTase genes BCAL3494 and BCAM0992 were deleted in both strains, and the phenotypes of the resulting deletion mutants were studied: deletion mutant ΔBCAL3494 showed changes in biofilm structure and cell aggregation, while ΔBCAM0992 was less motile. B. cenocepacia wild-type cultures treated with sinefungin, a known DNA MTase inhibitor, exhibited the same phenotype as DNA MTase deletion mutants. Single-molecule real-time sequencing was used to characterize the methylome of B. cenocepacia, including methylation at the origin of replication, and motifs CACAG and GTWWAC were identified as targets of BCAL3494 and BCAM0992, respectively. All genes with methylated motifs in their putative promoter region were identified, and qPCR experiments showed an upregulation of several genes, including biofilm- and motility-related genes, in MTase deletion mutants with unmethylated motifs, explaining the observed phenotypes in these mutants. In summary, our data confirm that DNA methylation plays an important role in regulating the expression of B. cenocepacia genes involved in biofilm formation, cell aggregation, and motility.IMPORTANCE CF patients diagnosed with Burkholderia cenocepacia infections often experience rapid deterioration of lung function, known as cepacia syndrome. B. cenocepacia has a large multireplicon genome, and much remains to be learned about regulation of gene expression in this organism. From studies in other (model) organisms, it is known that epigenetic changes through DNA methylation play an important role in this regulation. The identification of B. cenocepacia genes of which the expression is regulated by DNA methylation and identification of the regulatory systems involved in this methylation are likely to advance the biological understanding of B. cenocepacia cell adaptation via epigenetic regulation. In time, this might lead to novel approaches to tackle B. cenocepacia infections in CF patients.

Copper resistance genes of Burkholderia cenocepacia H111 identified by transposon sequencing.

Copper is an essential element but in excess is highly toxic and therefore cytoplasmic levels must be tightly controlled. Member of the genus Burkholderia are highly resistant to various heavy metals and are often isolated from acidic soils where copper bioavailability is high. In this study, we employed transposon sequencing (Tn-Seq) to identify copper resistance genes in Burkholderia cenocepacia H111. We identified a copper efflux system that shares similarities with the plasmid-based copper detoxification systems found in Escherichia coli and Pseudomonas syringae. We also found that several of the identified resistance determinants are involved in maintaining the integrity of the cell envelope, suggesting that proteins located in the outer membrane and periplasmic space are particularly sensitive to copper stress. Given that several of the resistance genes are required for the repair and turnover of misfolded proteins, we suggest that copper toxicity is caused by protein damage rather than by oxidative stress.

Patterns of Nucleotide Deletion and Insertion Inferred from Bacterial Pseudogenes.

Pseudogenes are a paradigm of neutral evolution and their study has the potential to reveal intrinsic mutational biases. However, this potential is mitigated by the fact that pseudogenes are quickly purged from bacterial genomes. Here, we assembled a large set of pseudogenes from genomes experiencing reductive evolution as well as functional references for which we could establish reliable phylogenetic relationships. Using this unique dataset, we identified 857 independent insertion and deletion mutations and discover a pervasive bias towards deletions, but not insertions, with sizes multiples of 3 nt. We further show that selective constraints for the preservation of gene frame are unlikely to account for the observed mutational bias and propose that a mechanistic bias in alternative end-joining repair, a recombination-independent double strand break DNA repair mechanism, is responsible for the accumulation of 3n deletions.

Leaf nodule symbiosis: function and transmission of obligate bacterial endophytes.

Various plant species establish intimate symbioses with bacteria within their aerial organs. The bacteria are contained within nodules or glands often present in distinctive patterns on the leaves, and have been used as taxonomic marker since the early 20th century. These structures are present in very diverse taxa, including dicots (Rubiaceae and Primulaceae) and monocots (Dioscorea). The symbionts colonize the plants throughout their life cycles and contribute bioactive secondary metabolites to the association. In this review, we present recent progress in the understanding of these plant-bacteria symbioses, including the modes of transmission, distribution and roles of the symbionts.

Heterologous Expression, Biosynthetic Studies, and Ecological Function of the Selective Gq-Signaling Inhibitor FR900359.

The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, is a strong and selective inhibitor of Gq proteins, making it an indispensable pharmacological tool to study Gq-related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as shown by in vivo experiments in mice, its affinity to insect Gq proteins, and insect toxicity studies. The uncultured endosymbiont of A. crenata was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. We here provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies, and the novel analogue AC-1, which demonstrates the flexibility of the FR starter condensation domains. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time.

The Essential Genome of Burkholderia cenocepacia H111.

The study of the minimum set of genes required to sustain life is a fundamental question in biological research. Recent studies on bacterial essential genes suggested that between 350 and 700 genes are essential to support autonomous bacterial cell growth. Essential genes are of interest as potential new antimicrobial drug targets; hence, our aim was to identify the essential genome of the cystic fibrosis (CF) isolate Burkholderia cenocepacia H111. Using a transposon sequencing (Tn-Seq) approach, we identified essential genes required for growth in rich medium under aerobic and microoxic conditions as well as in a defined minimal medium with citrate as a sole carbon source. Our analysis suggests that 398 genes are required for autonomous growth in rich medium, a number that represents only around 5% of the predicted genes of this bacterium. Five hundred twenty-six genes were required to support growth in minimal medium, and 434 genes were essential under microoxic conditions (0.5% O2). A comparison of these data sets identified 339 genes that represent the minimal set of essential genes required for growth under all conditions tested and can be considered the core essential genome of B. cenocepacia H111. The majority of essential genes were found to be located on chromosome 1, and few such genes were located on chromosome 2, where most of them were clustered in one region. This gene cluster is fully conserved in all Burkholderia species but is present on chromosome 1 in members of the closely related genus Ralstonia, suggesting that the transfer of these essential genes to chromosome 2 in a common ancestor contributed toward the separation of the two genera.IMPORTANCE Transposon sequencing (Tn-Seq) is a powerful method used to identify genes that are essential for autonomous growth under various conditions. In this study, we have identified a set of "core essential genes" that are required for growth under multiple conditions, and these genes represent potential antimicrobial targets. We also identified genes specifically required for growth under low-oxygen and nutrient-limited environments. We generated conditional mutants to verify the results of our Tn-Seq analysis and demonstrate that one of the identified genes was not essential per se but was an artifact of the construction of the mutant library. We also present verified examples of genes that were not truly essential but, when inactivated, showed a growth defect. These examples have identified so-far-underestimated shortcomings of this powerful method.

Evidence of horizontal gene transfer between obligate leaf nodule symbionts.

Bacteria of the genus Burkholderia establish an obligate symbiosis with plant species of the Rubiaceae and Primulaceae families. The bacteria, housed within the leaves, are transmitted hereditarily and have not yet been cultured. We have sequenced and compared the genomes of eight bacterial leaf nodule symbionts of the Rubiaceae plant family. All of the genomes exhibit features consistent with genome erosion. Genes potentially involved in the biosynthesis of kirkamide, an insecticidal C7N aminocyclitol, are conserved in most Rubiaceae symbionts. However, some have partially lost the kirkamide pathway due to genome erosion and are unable to synthesize the compound. Kirkamide synthesis is therefore not responsible for the obligate nature of the symbiosis. More importantly, we find evidence of intra-clade horizontal gene transfer (HGT) events affecting genes of the secondary metabolism. This indicates that substantial gene flow can occur at the early stages following host restriction in leaf nodule symbioses. We propose that host-switching events and plasmid conjugative transfers could have promoted these HGTs. This genomic analysis of leaf nodule symbionts gives, for the first time, new insights in the genome evolution of obligate symbionts in their early stages of the association with plants.

The genome analysis of Candidatus†Burkholderia crenata reveals that secondary metabolism may be a key function of the Ardisia crenata leaf nodule symbiosis.

A majority of Ardisia species harbour Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted hereditarily and have not yet been cultured outside of their host. Because the plants cannot develop beyond the seedling stage without their symbionts, the symbiosis is considered obligatory. We sequenced for the first time the genome of Candidatus Burkholderia crenata (Ca. B. crenata), the leaf nodule symbiont of Ardisia crenata. The genome of Ca. B. crenata is the smallest Burkholderia genome to date. It contains a large amount of insertion sequences and pseudogenes and displays features consistent with reductive genome evolution. The genome does not encode functions commonly associated with plant symbioses such as nitrogen fixation and plant hormone metabolism. However, we identified unique genes with a predicted role in secondary metabolism in the genome of Ca. B. crenata. Specifically, we provide evidence that the bacterial symbionts are responsible for the synthesis of compound FR900359, a cyclic depsipeptide with biomedical properties previously isolated from leaves of A. crenata.

Genome Sequence of Burkholderia cenocepacia H111, a Cystic Fibrosis Airway Isolate.

The Burkholderia cepacia complex (BCC) is a group of related bacterial species that are commonly isolated from environmental samples. Members of the BCC can cause respiratory infections in cystic fibrosis patients and immunocompromised individuals. We report here the genome sequence of Burkholderia cenocepacia H111, a well-studied model strain of the BCC.

Genetic Characterization of Legionella pneumophila Isolated from a Common Watershed in Comunidad Valenciana, Spain.

Legionella pneumophila infects humans to produce legionellosis and Pontiac fever only from environmental sources. In order to establish control measures and study the sources of outbreaks it is essential to know extent and distribution of strain variants of this bacterium in the environment. Sporadic and outbreak-related cases of legionellosis have been historically frequent in the Comunidad Valenciana region (CV, Spain), with a high prevalence in its Southeastern-most part (BV). Environmental investigations for the detection of Legionella pneumophila are performed in this area routinely. We present a population genetics study of 87 L. pneumophila strains isolated in 13 different localities of the BV area irrigated from the same watershed and compare them to a dataset of 46 strains isolated in different points of the whole CV. Our goal was to compare environmental genetic variation at two different geographic scales, at county and regional levels. Genetic diversity, recombination and population structure were analyzed with Sequence-Based Typing data and three intergenic regions. The results obtained reveal a low, but detectable, level of genetic differentiation between both datasets, mainly, but not only, attributed to the occurrence of unusual variants of the neuA locus present in the BV populations. This differentiation is still detectable when the 10 loci considered are analyzed independently, despite the relatively high incidence of the most common genetic variant in this species, sequence type 1 (ST-1). However, when the genetic data are considered without their associated geographic information, four major groups could be inferred at the genetic level which did not show any correlation with sampling locations. The overall results indicate that the population structure of these environmental samples results from the joint action of a global, widespread ST-1 along with genetic differentiation at shorter geographic distances, which in this case are related to the common watershed for the BV localities.

Evidence for diversifying selection in a set of Mycobacterium tuberculosis genes in response to antibiotic- and nonantibiotic-related pressure.

Tuberculosis (TB) is a global health problem estimated to kill 1.4 million people per year. Recent advances in the genomics of the causative agents of TB, bacteria known as the Mycobacterium tuberculosis complex (MTBC), have allowed a better comprehension of its population structure and provided the foundation for molecular evolution analyses. These studies are crucial for a better understanding of TB, including the variation of vaccine efficacy and disease outcome, together with the emergence of drug resistance. Starting from the analysis of 73 publicly available genomes from all the main MTBC lineages, we have screened for evidences of positive selection, a set of 576 genes previously associated with drug resistance or encoding membrane proteins. As expected, because antibiotics constitute strong selective pressure, some of the codons identified correspond to the position of confirmed drug-resistance-associated substitutions in the genes embB, rpoB, and katG. Furthermore, we identified diversifying selection in specific codons of the genes Rv0176 and Rv1872c coding for MCE1-associated transmembrane protein and a putative l-lactate dehydrogenase, respectively. Amino acid sequence analyses showed that in Rv0176, sites undergoing diversifying selection were in a predicted antigen region that varies between "modern" lineages and "ancient" MTBC/BCG strains. In Rv1872c, some of the sites under selection are predicted to impact protein function and thus might result from metabolic adaptation. These results illustrate that diversifying selection in MTBC is happening as a consequence of both antibiotic treatment and other evolutionary pressures.