Bacteria communicate via small diffusible molecules to mediate group-coordinated behavior, a process designated as quorum sensing. The basic molecular quorum sensing system of Gram-negative bacteria consists of a LuxI-type autoinducer synthase producing acyl-homoserine lactones (AHLs) as signaling molecules, and a LuxR-type receptor detecting the AHLs to control expression of specific genes. However, many proteobacteria possess one or more unpaired LuxR-type receptors that lack a cognate LuxI-like synthase, referred to as LuxR solos. The enteric and insect pathogenic bacteria of the genus Photorhabdus harbor an extraordinarily high number of LuxR solos, more than any other known bacteria, and all lack a LuxI-like synthase. Here, we focus on the presence and the different types of LuxR solos in the three known Photorhabdus species using bioinformatics analyses. Generally, the N-terminal signal-binding domain (SBD) of LuxR-type receptors sensing AHLs have a motif of six conserved amino acids that is important for binding and specificity of the signaling molecule. However, this motif is altered in the majority of the Photorhabdus-specific LuxR solos, suggesting the use of other signaling molecules than AHLs. Furthermore, all Photorhabdus species contain at least one LuxR solo with an intact AHL-binding motif, which might allow the ability to sense AHLs of other bacteria. Moreover, all three species have high AHL-degrading activity caused by the presence of different AHL-lactonases and AHL-acylases, revealing a high quorum quenching activity against other bacteria. However, the majority of the other LuxR solos in Photorhabdus have a N-terminal so-called PAS4-domain instead of an AHL-binding domain, containing different amino acid motifs than the AHL-sensors, which potentially allows the recognition of a highly variable range of signaling molecules that can be sensed apart from AHLs. These PAS4-LuxR solos are proposed to be involved in host sensing, and therefore in inter-kingdom signaling. Overall, Photorhabdus species are perfect model organisms to study bacterial communication via LuxR solos and their role for a symbiotic and pathogenic life style.

Biological diversity arises among genetically equal subpopulations in the same environment, a phenomenon called phenotypic heterogeneity. The life cycle of the enteric bacterium Photorhabdus luminescens involves a symbiotic interaction with nematodes as well as a pathogenic association with insect larvae. P. luminescens exists in two distinct phenotypic forms designated as primary (1°) and secondary (2°). In contrast to 1° cells, 2° cells are non-pigmented due to the absence of natural compounds, especially anthraquinones (AQs). We identified a novel type of transcriptional regulator, AntJ, which activates expression of the antA-I operon responsible for AQ production. AntJ heterogeneously activates the AQ production in single P. luminescens 1° cells, and blocks AQ production in 2° cells. AntJ contains a proposed ligand-binding WYL-domain, which is widespread among bacteria. AntJ is one of the rare examples of regulators that mediate heterogeneous gene expression by altering activity rather than copy number in single cells.

Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily.

The branched chain alcohol isobutanol exhibits superior physicochemical properties as an alternative biofuel. The yeast Saccharomyces cerevisiae naturally produces low amounts of isobutanol as a by-product during fermentations, resulting from the catabolism of valine. As S. cerevisiae is widely used in industrial applications and can easily be modified by genetic engineering, this microorganism is a promising host for the fermentative production of higher amounts of isobutanol.

In sequencing the genomes of two Xenorhabdus species, we encountered a large number of sequence repeats and assembly anomalies that stalled finishing efforts. This included a stretch of about 12 Kb that is over 99.9% identical between the plasmid and chromosome of X. nematophila.

Photorhabdus and Xenorhabdus are symbiotic with entomopathogenic nematodes (EPNs) of the genera Heterorhabditis and Steinernema, respectively. These bacteria produce several secondary metabolites including antimicrobial compounds. The objectives of this study were to isolate and identify EPNs and their symbiotic bacteria from Mae Wong National Park, Thailand and to evaluate the antibacterial activities of symbiont extracts against drug resistant bacteria. A total of 550 soil samples from 110 sites were collected between August 2014 and July 2015. A total of EPN isolates were obtained through baiting and White trap methods, which yielded 21 Heterorhabditis and 3 Steinernema isolates. Based on molecular identification and phylogenetic analysis, the most common species found in the present study was P. luminescens subsp. akhurstii associated with H. indica. Notably, two species of EPNs, H. zealandica and S. kushidai, and two species of symbiotic bacteria, X. japonica and P. temperata subsp. temperata represented new recorded organisms in Thailand. Furthermore, the association between P. temperata subsp. temperata and H. zealandica has not previously been reported worldwide. Disk diffusion, minimal inhibitory concentration, and minimal bactericidal concentration analyses demonstrated that the crude compound extracted by ethyl acetate from P. temperata subsp. temperata could inhibit the growth of up to 10 strains of drug resistant bacteria. Based on HPLC-MS analysis, compound classes in bacterial extracts were identified as GameXPeptide, xenoamicin, xenocoumacin, mevalagmapeptide phurealipids derivatives, and isopropylstilbene. Together, the results of this study provide evidence for the diversity of EPNs and their symbiotic bacteria in Mae Wong National Park, Thailand and demonstrate their novel associations. These findings also provide an important foundation for further research regarding the antimicrobial activity of Photorhabdus bacteria.

The interaction between the mammalian host and its resident gut microbiota is known to license adaptive immune responses. Nutritional constituents strongly influence composition and functional properties of the intestinal microbial communities. Here, we report that omission of a single essential amino acid - tryptophan - from the diet abrogates CNS autoimmunity in a mouse model of multiple sclerosis. Dietary tryptophan restriction results in impaired encephalitogenic T cell responses and is accompanied by a mild intestinal inflammatory response and a profound phenotypic shift of gut microbiota. Protective effects of dietary tryptophan restriction are abrogated in germ-free mice, but are independent of canonical host sensors of intracellular tryptophan metabolites. We conclude that dietary tryptophan restriction alters metabolic properties of gut microbiota, which in turn have an impact on encephalitogenic T cell responses. This link between gut microbiota, dietary tryptophan and adaptive immunity may help to develop therapeutic strategies for protection from autoimmune neuroinflammation.

Nonribosomal peptide synthetases (NRPSs) generate the core peptide scaffolds of many natural products. These include small cyclic dipeptides such as the insect feeding deterrent peramine, which is a pyrrolopyrazine (PPZ) produced by grass-endophytic Epichloë fungi. Biosynthesis of peramine is catalyzed by the 2-module NRPS, PpzA-1, which has a C-terminal reductase (R) domain that is required for reductive release and cyclization of the NRPS-tethered dipeptidyl-thioester intermediate. However, some PpzA variants lack this R domain due to insertion of a transposable element into the 3' end of ppzA We demonstrate here that these truncated PpzA variants utilize nonenzymatic cyclization of the dipeptidyl thioester to a 2,5-diketopiperazine (DKP) to synthesize a range of novel PPZ products. Truncation of the R domain is sufficient to subfunctionalize PpzA-1 into a dedicated DKP synthetase, exemplified by the truncated variant, PpzA-2, which has also evolved altered substrate specificity and reduced N-methyltransferase activity relative to PpzA-1. Further allelic diversity has been generated by recombination-mediated domain shuffling between ppzA-1 and ppzA-2, resulting in the ppzA-3 and ppzA-4 alleles, each of which encodes synthesis of a unique PPZ metabolite. This research establishes that efficient NRPS-catalyzed DKP biosynthesis can occur in vivo through nonenzymatic dipeptidyl cyclization and presents a remarkably clean example of NRPS evolution through recombinant exchange of functionally divergent domains. This work highlights that allelic variants of a single NRPS can result in a surprising level of secondary metabolite diversity comparable to that observed for some gene clusters.

Nonribosomal peptide synthetases (NRPSs) use terminal reductase domains for 2-electron reduction of the enzyme-bound thioester releasing the generated peptides as C-terminal aldehydes. Herein, we reveal the biosynthesis of a pyrazine that originates from an aldehyde-generating minimal NRPS termed ATRed in entomopathogenic Xenorhabdus indica. Reductase domains were also investigated in terms of NRPS engineering and, although no general applicable approach was deduced, we show that they can indeed be used for the production of similar natural and unnatural pyrazinones.

Non-ribosomal peptide synthetases (NRPSs) are large multienzyme machineries. They synthesize numerous important natural products starting from amino acids. For peptide synthesis functionally specialized NRPS modules interact in a defined manner. Individual modules are either located on a single or on multiple different polypeptide chains. The "peptide-antimicrobial-Xenorhabdus" (PAX) peptide producing NRPS PaxS from Xenorhabdus bacteria consists of the three proteins PaxA, PaxB and PaxC. Different docking domains (DDs) located at the N-termini of PaxB and PaxC and at the C-termini of PaxA and BaxB mediate specific non-covalent interactions between them. The N-terminal docking domains precede condensation domains while the C-terminal docking domains follow thiolation domains. The binding specificity of individual DDs is important for the correct assembly of multi-protein NRPS systems. In many multi-protein NRPS systems the docking domains are sufficient to mediate the necessary interactions between individual protein chains. However, it remains unclear if this is a general feature for all types of structurally different docking domains or if the neighboring domains in some cases support the function of the docking domains. Here, we report the 1H, 13C and 15 N NMR resonance assignments for a C-terminal di-domain construct containing a thiolation (T) domain followed by a C-terminal docking domain (CDD) from PaxA and for its binding partner - the N-terminal docking domain (NDD) from PaxB from the Gram-negative entomopathogenic bacterium Xenorhabdus cabanillasii JM26 in their free states and for a 1:1 complex formed by the two proteins. These NMR resonance assignments will facilitate further structural and dynamic studies of this protein complex.

The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study, we conducted genome mining to explore the biosynthetic potential of entomopathogenic bacteria belonging to the genera Xenorhabdus and Photorhabdus. By utilizing next-generation sequencing and bioinformatics tools, we identified novel biosynthetic gene clusters (BGCs) in the genomes of the bacteria, specifically plu00736 and plu00747. These clusters were identified as unidentified non-ribosomal peptide synthetase (NRPS) and unidentified type I polyketide synthase (T1PKS) clusters. These BGCs exhibited unique genetic architecture and encoded several putative enzymes and regulatory elements, suggesting its involvement in the synthesis of bioactive secondary metabolites. Furthermore, comparative genome analysis revealed that these BGCs were distinct from previously characterized gene clusters, indicating the potential for the production of novel compounds. Our findings highlighted the importance of genome mining as a powerful approach for the discovery of biosynthetic gene clusters and the identification of novel bioactive compounds. Further investigations involving expression studies and functional characterization of the identified BGCs will provide valuable insights into the biosynthesis and potential applications of these bioactive compounds.

An entomopathogenic bacterium, Photorhabdus temperata subsp. temperata, is mutualistic to its host nematode, Heterorhabditis megidis. The infective juvenile nematodes enter target insects through natural openings and release the symbiotic bacteria into the insect hemocoel. The released bacteria suppress the insect immune responses and cause septicemia through their secondary metabolites. GameXPeptide (GXP) is one of the common secondary metabolites of most Photorhabdus species and is produced by the catalytic activity of a specific non-ribosomal peptide synthetase called GxpS encoded by the gxpS gene. This study confirmed gxpS to be encoded in the P. temperata temperata genome and analyzed its expression during bacterial growth. LC-MS/MS analysis of the bacterial culture broth contained at least four different GXPs (GXP-A to GXP-D), in which GXP-A was the most abundant. To investigate GXP synthesis following gxpS expression, the gxpS promoter of P. temperata temperata was replaced with an inducible arabinose promoter by homologous recombination. The gxpS transcript levels in the mutant were altered by the addition of l-arabinose. Without the inducer, the gxpS transcript level was significantly lower compared to the wild type and produced significantly lower amounts of the four GXPs. The addition of the inducer to the mutant significantly increased gxpS expression and produced significantly higher levels of the four GXPs compared to the wild type. The metabolite extracts obtained from wild-type and mutant bacteria showed differential immunosuppressive activities according to their GXP contents against the cellular and humoral immune responses of a lepidopteran insect, Spodoptera exigua. Interestingly, the gxpS-mutant bacteria showed less insecticidal activity compared to the wild type, whereas the addition of GXP to the mutant significantly restored insecticidal activity. These results suggest that the gxpS gene encoded in P. temperata temperata is responsible for the production of at least four different GXPs, which play crucial roles in bacterial virulence.

Photorhabdus are highly effective insect pathogenic bacteria that exist in a mutualistic relationship with Heterorhabditid nematodes. Unlike other members of the genus, Photorhabdus asymbiotica can also infect humans. Most Photorhabdus cannot replicate above 34°C, limiting their host-range to poikilothermic invertebrates. In contrast, P. asymbiotica must necessarily be able to replicate at 37°C or above. Many well-studied mammalian pathogens use the elevated temperature of their host as a signal to regulate the necessary changes in gene expression required for infection. Here we use RNA-seq, proteomics and phenotype microarrays to examine temperature dependent differences in transcription, translation and phenotype of P. asymbiotica at 28°C versus 37°C, relevant to the insect or human hosts respectively. Our findings reveal relatively few temperature dependant differences in gene expression. There is however a striking difference in metabolism at 37°C, with a significant reduction in the range of carbon and nitrogen sources that otherwise support respiration at 28°C. We propose that the key adaptation that enables P. asymbiotica to infect humans is to aggressively acquire amino acids, peptides and other nutrients from the human host, employing a so called "nutritional virulence" strategy. This would simultaneously cripple the host immune response while providing nutrients sufficient for reproduction. This might explain the severity of ulcerated lesions observed in clinical cases of Photorhabdosis. Furthermore, while P. asymbiotica can invade mammalian cells they must also resist immediate killing by humoral immunity components in serum. We observed an increase in the production of the insect Phenol-oxidase inhibitor Rhabduscin normally deployed to inhibit the melanisation immune cascade. Crucially we demonstrated this molecule also facilitates protection against killing by the alternative human complement pathway.

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.

Bacteria from the Bacteroidetes phylum are known producers of the chemotaxonomic relevant flexirubins. These orange pigments comprise a non-isoprenoid aryl-polyene carboxylic acid esterified with a dialkylresorcinol. Herein, we report a gene cluster from Chitinophaga pinensis encoding the biosynthesis of the polyene moiety and the biochemical characterization of a tyrosine ammonia-lyase and a 4-coumarate-CoA ligase responsible for the initiation of the polyene biosynthesis. Additionally, the flexirubin of C. pinensis was characterized by a combination of feeding experiments, high-performance liquid chromatography tandem mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry.

In the last decades, natural products from lichens have gained more interest for pharmaceutical application due to the broad range of their biological activity. However, isolation of the compounds of interest directly from the lichen is neither feasible nor sustainable due to slow growth of many lichens. In order to develop a pipeline for heterologous expression of lichen biosynthesis gene clusters and thus the sustainable production of their bioactive compounds we have identified and characterized the phosphopantheteinyl transferase (PPTase) EppA from the lichen Evernia prunastri. The Sfp-type PPTase EppA was functionally characterized through heterologous expression in E. coli using the production of the blue pigment indigoidine as readout and by complementation of a lys5 deletion in S. cerevisiae.

Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes.

The synthesis of the recently characterized depsipeptide szentiamide (1), which is produced by the entomopathogenic bacterium Xenorhabdus szentirmaii, is described. Whereas no biological activity was previously identified for 1, the material derived from the efficient synthesis enabled additional bioactivity tests leading to the identification of a notable activity against insect cells and Plasmodium falciparum, the causative agent of malaria.

Xenorhabdus and Photorhabdus can produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial activity of Xenorhabdus and Photorhabdus against 15 antibiotic-resistant bacteria strains. Photorhabdus extracts had strong inhibitory the growth of Methicillin-resistant Staphylococcus aureus (MRSA) by disk diffusion. The P. akhurstii s subsp. akhurstii (bNN168.5_TH) extract showed lower minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The interaction between either P. akhurstii subsp. akhurstii (bNN141.3_TH) or P. akhurstii subsp. akhurstii (bNN168.5_TH) or P. hainanensis (bNN163.3_TH) extract in combination with oxacillin determined by checkerboard assay exhibited partially synergistic interaction with fractional inhibitory concentration index (FICI) of 0.53. Time-killing assay for P. akhurstii subsp. akhurstii (bNN168.5_TH) extract against S. aureus strain PB36 significantly decreased cell viability from 105 CFU/ml to 103 CFU/ml within 30 min (P < 0.001, t-test). Transmission electron microscopic investigation elucidated that the bNN168.5_TH extract caused treated S. aureus strain PB36 (MRSA) cell membrane damage. The biosynthetic gene clusters of the bNN168.5_TH contained non-ribosomal peptide synthetase cluster (NRPS), hybrid NRPS-type l polyketide synthase (PKS) and siderophore, which identified potentially interesting bioactive products: xenematide, luminmide, xenortide A-D, luminmycin A, putrebactin/avaroferrin and rhizomide A-C. This study demonstrates that bNN168.5_TH showed antibacterial activity by disrupting bacterial cytoplasmic membrane and the draft genome provided insights into the classes of bioactive products. This also provides a potential approach in developing a novel antibacterial agent.

Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution.