Insight in the quorum sensing-driven lifestyle of the non-pathogenic Agrobacterium tumefaciens 6N2 and the interactions with the yeast Meyerozyma guilliermondii.

Agrobacterium tumefaciens is considered a prominent phytopathogen, though most isolates are nonpathogenic. Agrobacteria can inhabit plant tissues interacting with other microorganisms. Yeasts are likewise part of these communities. We analyzed the quorum sensing (QS) systems of A. tumefaciens strain 6N2, and its relevance for the interaction with the yeast Meyerozyma guilliermondii, both sugarcane endophytes. We show that strain 6N2 is nonpathogenic, produces OHC8-HSL, OHC10-HSL, OC12-HSL and OHC12-HSL as QS signals, and possesses a complex QS architecture, with one truncated, two complete systems, and three additional QS-signal receptors. A proteomic approach showed differences in QS-regulated proteins between pure (64 proteins) and dual (33 proteins) cultures. Seven proteins were consistently regulated by quorum sensing in pure and dual cultures. M. guilliermondii proteins influenced by QS activity were also evaluated. Several up- and down- regulated proteins differed depending on the bacterial QS. These results show the QS regulation in the bacteria-yeast interactions.

Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme.

Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies.

Modifications in the pmrB gene are the primary mechanism for the development of chromosomally encoded resistance to polymyxins in uropathogenic Escherichia coli.

Objectives: Polymyxins remain one of the last-resort drugs to treat infections caused by MDR Gram-negative pathogens. Here, we determined the mechanisms by which chromosomally encoded resistance to colistin and polymyxin B can arise in the MDR uropathogenic Escherichia coli ST131 reference strain EC958. Methods: Two complementary approaches, saturated transposon mutagenesis and spontaneous mutation induction with high concentrations of colistin and polymyxin B, were employed to select for mutations associated with resistance to polymyxins. Mutants were identified using transposon-directed insertion-site sequencing or Illumina WGS. A resistance phenotype was confirmed by MIC and further investigated using RT-PCR. Competitive growth assays were used to measure fitness cost. Results: A transposon insertion at nucleotide 41 of the pmrB gene (EC958pmrB41-Tn5) enhanced its transcript level, resulting in a 64- and 32-fold increased MIC of colistin and polymyxin B, respectively. Three spontaneous mutations, also located within the pmrB gene, conferred resistance to both colistin and polymyxin B with a corresponding increase in transcription of the pmrCAB genes. All three mutations incurred a fitness cost in the absence of colistin and polymyxin B. Conclusions: This study identified the pmrB gene as the main chromosomal target for induction of colistin and polymyxin B resistance in E. coli.

Transfer of the potato plant isolates of Pectobacterium wasabiae to Pectobacterium parmentieri sp. nov.

Pectobacterium wasabiae was originally isolated from Japanese horseradish (Eutrema wasabi), but recently some Pectobacterium isolates collected from potato plants and tubers displaying blackleg and soft rot symptoms were also assigned to P. wasabiae. Here, combining genomic and phenotypical data, we re-evaluated their taxonomic position. PacBio and Illumina technologies were used to complete the genome sequences of P. wasabiae CFBP 3304T and RNS 08-42-1A. Multi-locus sequence analysis showed that the P. wasabiae strains RNS 08-42-1A, SCC3193, CFIA1002 and WPP163, which were collected from potato plant environment, constituted a separate clade from the original Japanese horseradish P. wasabiae. The taxonomic position of these strains was also supported by calculation of the in-silico DNA-DNA hybridization, genome average nucleotide indentity, alignment fraction and average nucleotide indentity values. In addition, they were phenotypically distinguished from P. wasabiae strains by producing acids from (+)-raffinose, α-d(+)-α-lactose, d(+)-galactose and (+)-melibiose but not from methyl α-d-glycopyranoside, (+)-maltose or malonic acid. The name Pectobacterium parmentieri sp. nov. is proposed for this taxon; the type strain is RNS 08-42-1AT (=CFBP 8475T=LMG 29774T).

Molecular analysis of asymptomatic bacteriuria Escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition.

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.

Population genomics reveals additive and replacing horizontal gene transfers in the emerging pathogen Dickeya solani.

BACKGROUND: Dickeya solani is an emerging pathogen that causes soft rot and blackleg diseases in several crops including Solanum tuberosum, but little is known about its genomic diversity and evolution. RESULTS: We combined Illumina and PacBio technologies to complete the genome sequence of D. solani strain 3337 that was used as a reference to compare with 19 other genomes (including that of the type strain IPO2222(T)) which were generated by Illumina technology. This population genomic analysis highlighted an unexpected variability among D. solani isolates since it led to the characterization of two distinct sub-groups within the D. solani species. This approach also revealed different types of variations such as scattered SNP/InDel variations as well as replacing and additive horizontal gene transfers (HGT). Infra-species (between the two D. solani sub-groups) and inter-species (between D. solani and D. dianthicola) replacing HGTs were observed. Finally, this work pointed that genetic and functional variation in the motility trait could contribute to aggressiveness variability in D. solani. CONCLUSIONS: This work revealed that D. solani genomic variability may be caused by SNPs/InDels as well as replacing and additive HGT events, including plasmid acquisition; hence the D. solani genomes are more dynamic than that were previously proposed. This work alerts on precautions in molecular diagnosis of this emerging pathogen.

Core genome and plasmidome of the quorum-quenching bacterium Rhodococcus erythropolis.

Rhodococcus erythropolis is a worldwide-distributed actinobacterium that exhibits a remarkable metabolic versatility illustrated by its ability to degrade complex compounds, such as quorum-sensing signals N-acylhomoserine lactones (NAHLs), phenols, sterols and fuel derivatives. Because of its catabolic properties, R. erythropolis strains are proposed as anti-biofouling agents against NAHL-dependent biofilms, biocontrol agents against NAHL-emitting plant pathogens, and bioremediation agents in contaminated waters and soils. Here, we used the PacBio technology to resolve the complete genome sequence of the biocontrol strain R. erythropolis R138. Its genome consisted in a circular chromosome (6,236,862 bp), a linear plasmid pLRE138 (477,915 bp) and a circular plasmid pCRE138 (91,729 bp). In addition, draft genomes of five R. erythropolis strains were determined by Illumina technology and compared with the other five R. erythropolis genomes that are available in public databases: 5,825 common CDSs were present in all of the eleven analyzed genomes and represented up to 87 % of those identified in R. erythropolis R138. This study highlighted the high proportion of core-genome genes in R. erythropolis, but a high variability of the plasmid content. Key-metabolic pathways which are involved in the degradation of complex molecules, such as NAHLs and phenol, catechol and sterol derivatives are coded by the R. erythropolis core-genome.

Insights from the genome sequence of quorum-quenching Staphylococcus sp. strain AL1, isolated from traditional Chinese soy sauce brine fermentation.

We report the draft genome sequence of Staphylococcus sp. strain AL1, which degrades quorum-sensing molecules (namely, N-acyl homoserine lactones). To the best of our knowledge, this is the first documentation that reports the whole genome sequence and quorum-quenching activity of Staphylococcus sp. strain AL1.

Heavy-metal resistance of a France vineyard soil bacterium, Pseudomonas mendocina strain S5.2, revealed by whole-genome sequencing.

Here we present the draft genome of Pseudomonas mendocina strain S5.2, possessing tolerance to a high concentration of copper. In addition to being copper resistant, the genome of P. mendocina strain S5.2 contains a number of heavy-metal-resistant genes known to confer resistance to multiple heavy-metal ions.

Characterization of quorum sensing and quorum quenching soil bacteria isolated from Malaysian tropical montane forest.

We report the production and degradation of quorum sensing N-acyl-homoserine lactones by bacteria isolated from Malaysian montane forest soil. Phylogenetic analysis indicated that these isolates clustered closely to the genera of Arthrobacter, Bacillus and Pseudomonas. Quorum quenching activity was detected in six isolates of these three genera by using a series of bioassays and rapid resolution liquid chromatography analysis. Biosensor screening and high resolution liquid chromatography-mass spectrometry analysis revealed the production of N-dodecanoyl-L-homoserine lactone (C12-HSL) by Pseudomonas frederiksbergensis (isolate BT9). In addition to degradation of a wide range of N-acyl-homoserine lactones, Arthrobacter and Pseudomonas spp. also degraded p-coumaroyl-homoserine lactone. To the best of our knowledge, this is the first documentation of Arthrobacter and Pseudomonas spp. capable of degrading p-coumaroyl-homoserine lactone and the production of C12-HSL by P. frederiksbergensis.

Integrating multiple genomic technologies to investigate an outbreak of carbapenemase-producing Enterobacter hormaechei.

Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we report the application of several complementary whole-genome sequencing (WGS) technologies to characterise a hospital outbreak of blaIMP-4 carbapenemase-producing E. hormaechei. Using Illumina sequencing, we determined that all outbreak strains were sequence type 90 (ST90) and near-identical. Comparison to publicly available data linked all outbreak isolates to a 2013 isolate from the same ward, suggesting an environmental source in the hospital. Using Pacific Biosciences sequencing, we resolved the complete context of the blaIMP-4 gene on a large IncHI2 plasmid carried by all IMP-4-producing strains across different hospitals. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing. Finally, Oxford Nanopore sequencing rapidly resolved the true relationship of subsequent isolates to the initial outbreak. Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak.

Genomic analyses of two Alteromonas stellipolaris strains reveal traits with potential biotechnological applications.

The Alteromonas stellipolaris strains PQQ-42 and PQQ-44, previously isolated from a fish hatchery, have been selected on the basis of their strong quorum quenching (QQ) activity, as well as their ability to reduce Vibrio-induced mortality on the coral Oculina patagonica. In this study, the genome sequences of both strains were determined and analyzed in order to identify the mechanism responsible for QQ activity. Both PQQ-42 and PQQ-44 were found to degrade a wide range of N-acylhomoserine lactone (AHL) QS signals, possibly due to the presence of an aac gene which encodes an AHL amidohydrolase. In addition, the different colony morphologies exhibited by the strains could be related to the differences observed in genes encoding cell wall biosynthesis and exopolysaccharide (EPS) production. The PQQ-42 strain produces more EPS (0.36 g l-1) than the PQQ-44 strain (0.15 g l-1), whose chemical compositions also differ. Remarkably, PQQ-44 EPS contains large amounts of fucose, a sugar used in high-value biotechnological applications. Furthermore, the genome of strain PQQ-42 contained a large non-ribosomal peptide synthase (NRPS) cluster with a previously unknown genetic structure. The synthesis of enzymes and other bioactive compounds were also identified, indicating that PQQ-42 and PQQ-44 could have biotechnological applications.

High-quality Schistosoma haematobium genome achieved by single-molecule and long-range sequencing.

BACKGROUND: Schistosoma haematobium causes urogenital schistosomiasis, a neglected tropical disease affecting >100 million people worldwide. Chronic infection with this parasitic trematode can lead to urogenital conditions including female genital schistosomiasis and bladder cancer. At the molecular level, little is known about this blood fluke and the pathogenesis of the disease that it causes. To support molecular studies of this carcinogenic worm, we reported a draft genome for S. haematobium in 2012. Although a useful resource, its utility has been somewhat limited by its fragmentation. FINDINGS: Here, we systematically enhanced the draft genome of S. haematobium using a single-molecule and long-range DNA-sequencing approach. We achieved a major improvement in the accuracy and contiguity of the genome assembly, making it superior or comparable to assemblies for other schistosome species. We transferred curated gene models to this assembly and, using enhanced gene annotation pipelines, inferred a gene set with as many or more complete gene models as those of other well-studied schistosomes. Using conserved, single-copy orthologs, we assessed the phylogenetic position of S. haematobium in relation to other parasitic flatworms for which draft genomes were available. CONCLUSIONS: We report a substantially enhanced genomic resource that represents a solid foundation for molecular research on S. haematobium and is poised to better underpin population and functional genomic investigations and to accelerate the search for new disease interventions.

Population dynamics of an Escherichia coli ST131 lineage during recurrent urinary tract infection.

Recurrent urinary tract infections (rUTIs) are extremely common, with ~ 25% of all women experiencing a recurrence within 1 year of their original infection. Escherichia coli ST131 is a globally dominant multidrug resistant clone associated with high rates of rUTI. Here, we show the dynamics of an ST131 population over a 5-year period from one elderly woman with rUTI since the 1970s. Using whole genome sequencing, we identify an indigenous clonal lineage (P1A) linked to rUTI and persistence in the fecal flora, providing compelling evidence of an intestinal reservoir of rUTI. We also show that the P1A lineage possesses substantial plasmid diversity, resulting in the coexistence of antibiotic resistant and sensitive intestinal isolates despite frequent treatment. Our longitudinal study provides a unique comprehensive genomic analysis of a clonal lineage within a single individual and suggests a population-wide resistance mechanism enabling rapid adaptation to fluctuating antibiotic exposure.

Complete Chromosome and Plasmid Sequences of Two Plant Pathogens, Dickeya solani Strains D s0432-1 and PPO 9019.

Dickeya solani species are emerging bacterial pathogens of Solanum tuberosum Here, we announce the complete genome sequences of two strains, Dickeya solani D s0432-1 and PPO 9019. Strain PPO 9019 represents the first described member of the genus Dickeya with an extrachromosomal genetic element.

Complete Genome Sequences of the Plant Pathogens Dickeya solani RNS 08.23.3.1.A and Dickeya dianthicola RNS04.9.

Dickeya spp. are bacterial pathogens causing soft-rot and blackleg diseases on a wide range of ornamental plants and crops. In this paper, we announce the PacBio complete genome sequences of the plant pathogens Dickeya solani RNS 08.23.3.1.A (PRI3337) and Dickeya dianthicola RNS04.9.

Complete Genome Sequence of Streptomyces sp. TN58, a Producer of Acyl Alpha-l-Rhamnopyranosides.

Streptomyces sp. TN58, isolated from a Tunisian soil sample, produces several natural products, including acyl alpha-l-rhamnopyranosides. It possesses a 7.6-Mb linear chromosome. This is, to our knowledge, the first genome sequence of a microorganism known to produce acyl alpha-l-rhamnopyranosides, and it will be helpful to study the biosynthesis of these specialized metabolites.

Phenotypic and genomic survey on organic acid utilization profile of Pseudomonas mendocina strain S5.2, a vineyard soil isolate.

Root exudates are chemical compounds that are released from living plant roots and provide significant energy, carbon, nitrogen and phosphorus sources for microbes inhabiting the rhizosphere. The exudates shape the microflora associated with the plant, as well as influences the plant health and productivity. Therefore, a better understanding of the trophic link that is established between the plant and the associated bacteria is necessary. In this study, a comprehensive survey on the utilization of grapevine and rootstock related organic acids were conducted on a vineyard soil isolate which is Pseudomonas mendocina strain S5.2. Phenotype microarray analysis has demonstrated that this strain can utilize several organic acids including lactic acid, succinic acid, malic acid, citric acid and fumaric acid as sole growth substrates. Complete genome analysis using single molecule real-time technology revealed that the genome consists of a 5,120,146 bp circular chromosome and a 252,328 bp megaplasmid. A series of genetic determinants associated with the carbon utilization signature of the strain were subsequently identified in the chromosome. Of note, the coexistence of genes encoding several iron-sulfur cluster independent isoenzymes in the genome indicated the importance of these enzymes in the events of iron deficiency. Synteny and comparative analysis have also unraveled the unique features of D-lactate dehydrogenase of strain S5.2 in the study. Collective information of this work has provided insights on the metabolic role of this strain in vineyard soil rhizosphere.

Genome-Wide Discovery of Genes Required for Capsule Production by Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes (mprA and lrhA) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production.IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival and persistence during infection. The K1 serotype is one example of a polysaccharide capsule type and is strongly associated with UPEC strains that cause UTIs, bloodstream infections, and meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily increasing, highlighting the need to better understand factors (e.g., the capsule) that contribute to UPEC pathogenesis. This study describes the original and novel application of lytic capsule-specific phage killing, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing to define the entire complement of genes required for capsule production in UPEC. Our comprehensive approach uncovered new genes involved in the regulation of this key virulence determinant.