Multicenter assessment of shotgun metagenomics for pathogen detection.

BACKGROUND: Shotgun metagenomics has been used clinically for diagnosing infectious diseases. However, most technical assessments have been limited to individual sets of reference standards, experimental workflows, and laboratories. METHODS: A reference panel and performance metrics were designed and used to examine the performance of shotgun metagenomics at 17 laboratories in a coordinated collaborative study. We comprehensively assessed the reliability, key performance determinants, reproducibility, and quantitative potential. FINDINGS: Assay performance varied significantly across sites and microbial classes, with a read depth of 20 millions as a generally cost-efficient assay setting. Results of mapped reads by shotgun metagenomics could indicate relative and intra-site (but not absolute or inter-site) microbial abundance. INTERPRETATION: Assay performance was significantly impacted by the microbial type, the host context, and read depth, which emphasizes the importance of these factors when designing reference reagents and benchmarking studies. Across sites, workflows and platforms, false positive reporting and considerable site/library effects were common challenges to the assay's accuracy and quantifiability. Our study also suggested that laboratory-developed shotgun metagenomics tests for pathogen detection should aim to detect microbes at 500 CFU/mL (or copies/mL) in a clinically relevant host context (10^5 human cells/mL) within a 24h turn-around time, and with an efficient read depth of 20M. FUNDING: This work was supported by National Science and Technology Major Project of China (2018ZX10102001).

LuxR Solos in the Plant Endophyte Kosakonia sp. Strain KO348.

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.

Molecular Mechanisms of Phosphate Stress Activation of Pseudomonas aeruginosa Quorum Sensing Systems.

The hierarchical quorum sensing (QS) systems of Pseudomonas aeruginosa, consisting of las, pqs, and rhl, coordinate the expression of bacterial virulence genes. Previous studies showed that under phosphate deficiency conditions, two-component regulatory system PhoRB could activate various genes involved in cytotoxicity through modulation of QS systems, but the mechanism by which PhoR/PhoB influences QS remains largely unknown. Here, we provide evidence that among the key QS regulatory genes in P. aeruginosa, rhlR, pqsA, mvfR, and lasI were activated by the response regulator PhoB under phosphate-depleted conditions. We show that PhoB is a strong competitor against LasR and RsaL for binding to the promoter of lasI and induces significant expression of lasI, rhlR, and mvfR. However, expression of lasI, encoding the signal 3-oxo-C12-HSL, was increased only marginally under the same phosphate-depleted conditions. This seeming inconsistency was attributed to the induction of pvdQ, which encodes an enzyme for degradation of 3-oxo-C12-HSL signal molecules. Taken together, the results from this study demonstrate that through the two-component regulatory system PhoR/PhoB, phosphate depletion stress could influence the QS network by modulating several key regulators, including lasI, rhlR, mvfR, and pvdQ The findings highlight not only the potency of the PhoR/PhoB-mediated bacterial stress response mechanism but also the plasticity of the P. aeruginosa QS systems in coping with the changed environmental conditions.IMPORTANCE It is not fully understood how phosphate deficiency could influence the virulence of Pseudomonas aeruginosa through modulation of the bacterial QS systems. This report presents a systemic investigation on the impact of phosphate depletion on the hierarchy of quorum sensing systems of P. aeruginosa The results showed that phosphate stress could have an extensive impact on the QS networks of this bacterial pathogen. Among the 7 QS regulatory genes representing the 3 sets of QS systems tested, 4 were significantly upregulated by phosphate depletion stress through the PhoR/PhoB two-component regulatory system, especially the upstream QS regulatory gene lasI We also present evidence that the response regulator PhoB was a strong competitor against the las regulators LasR and RsaL for the lasI promoter, unveiling the mechanistic basis of the process by which phosphate stress could modulate the bacterial QS systems.

Quorum Sensing in Pseudomonas savastanoi pv. savastanoi and Erwinia toletana: Role in Virulence and Interspecies Interactions in the Olive Knot.

The olive knot disease (Olea europea L.) is caused by the bacterium Pseudomonas savastanoi pv. savastanoi. P. savastanoi pv. savastanoi in the olive knot undergoes interspecies interactions with the harmless endophyte Erwinia toletana; P. savastanoi pv. savastanoi and E. toletana colocalize and form a stable community, resulting in a more aggressive disease. P. savastanoi pv. savastanoi and Etoletana produce the same type of the N-acylhomoserine lactone (AHL) quorum sensing (QS) signal, and they share AHLs in planta In this work, we have further studied the AHL QS systems of P. savastanoi pv. savastanoi and Etoletana in order to determine possible molecular mechanism(s) involved in this bacterial interspecies interaction/cooperation. The AHL QS regulons of P. savastanoi pv. savastanoi and Etoletana were determined, allowing the identification of several QS-regulated genes. Surprisingly, the P. savastanoi pv. savastanoi QS regulon consisted of only a few loci whereas in Etoletana many putative metabolic genes were regulated by QS, among which are several involved in carbohydrate metabolism. One of these loci was the aldolase-encoding gene garL, which was found to be essential for both colocalization of P. savastanoi pv. savastanoi and Etoletana cells inside olive knots as well as knot development. This study further highlighted that pathogens can cooperate with commensal members of the plant microbiome.IMPORTANCE This is a report on studies of the quorum sensing (QS) systems of the olive knot pathogen Pseudomonas savastanoi pv. savastanoi and olive knot cooperator Erwinia toletana These two bacterial species form a stable community in the olive knot, share QS signals, and cooperate, resulting in a more aggressive disease. In this work we further studied the QS systems by determining their regulons as well as by studying QS-regulated genes which might play a role in this cooperation. This represents a unique in vivo interspecies bacterial virulence model and highlights the importance of bacterial interspecies interaction in disease.

Studies on synthetic LuxR solo hybrids.

A sub-group of LuxR family of proteins that plays important roles in quorum sensing, a process of cell-cell communication, is widespread in proteobacteria. These proteins have a typical modular structure consisting of N-ter autoinducer binding and C-ter helix-turn-helix (HTH) DNA binding domains. The autoinducer binding domain recognizes signaling molecules which are most often N-acyl homoserine lactones (AHLs) but could also be other novel and yet unidentified molecules. In this study we carried out a series of specific domain swapping and promoter activation experiments as a first step to engineer synthetic signaling modules, taking advantage of the modularity and the versatile/diverse signal specificities of LuxR proteins. In our experiments the N-ter domains from different LuxR homologs were either interchanged or placed in tandem followed by a C-ter domain. The rational design of the hybrid proteins was supported by a structure-based homology modeling studies of three members of the LuxR family (i.e., LasR, RhlR, and OryR being chosen for their unique ligand binding specificities) and of selected chimeras. Our results reveal that these LuxR homologs were able to activate promoter elements that were not their usual targets; we also show that hybrid LuxR proteins retained the ability to recognize the signal specific for their N- ter autoinducer binding domain. However, the activity of hybrid LuxR proteins containing two AHL binding domains in tandem appears to depend on the organization and nature of the introduced domains. This study represents advances in the understanding of the modularity of LuxR proteins and provides additional possibilities to use hybrid proteins in both basic and applied synthetic biology based research.

Draft Genome Sequence of Rice Endophyte-Associated Isolate Kosakonia oryzae KO348.

Kosakonia oryzae KO348 is an endophytic and plant growth-promoting strain isolated from the roots of rice in Italy. Here, we report the draft genome sequence of Kosakonia oryzae KO348.

Colonization by endophytic Ochrobactrum anthropi†Mn1 promotes growth of Jerusalem artichoke.

The Ochrobactrum anthropi Mn1 strain, taxonomically identified using 16S ribosomal DNA sequence, was isolated from roots of Jerusalem artichoke. Its endophytic colonization was investigated microscopically using green fluorescent protein introduced by vector pHC60. The strain entered Jerusalem artichoke tissues through the root, and was localized in the roots and stems. The plant growth-promoting (PGP) effects of O. anthropi Mn1 were assessed in greenhouse as well as field trials with different nitrogen supplies. Only under moderate to ample nitrogen supply, could O. anthropi Mn1 promoted growth of host plant. The PGP effects of the strain were symbiotic nitrogen fixation, root morphological optimization and enhanced nutrient uptake. We hypothesize that the symbiotic interspecies interaction might be quorum sensing related.

Influence of nitrogen fertilization on diazotrophic communities in the rhizosphere of the Jerusalem artichoke (Helianthus tuberosus L.).

Diazotrophs in the soil may be influenced by plant factors as well as nitrogen (N) fertilization. In this study, we investigated potential diazotrophic communities in the rhizosphere of the Jerusalem artichoke (Helianthus tuberosus L.) supplied with differing amounts of N. The community structure of N(2)-fixing bacteria was profiled using the length heterogeneity polymerase chain reaction (LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP) based on a variation in the nifH gene. Higher numbers of diazotrophs were detected by T-RFLP compared to LH-PCR. The lowest number of N(2)-fixing bacteria was observed in the rhizosphere soil with high N fertilization. T-RFLP was a better method than LH-PCR for profiling microbial diversity of diazotrophs using multidimensional scaling (MDS) and analysis of similarity (ANOSIM) of fingerprints as well as diversity measures. The supply of N fertilizer appeared to negatively influence the abundance of diazotrophs in the rhizophere of the Jerusalem artichoke.