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September 22, 2019

Functional and genome sequence-driven characterization of tal effector gene repertoires reveals novel variants with altered specificities in closely related Malian Xanthomonas oryzae pv. oryzae strains.

Rice bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years, BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TalF (formerly Tal5) or TalC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that talF and talC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either talC- or talF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TalF and TalC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related tal effector genes. Notably, a new TalF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TalB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TalB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in tal repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa.

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September 22, 2019

Detection and characterization of a clinical Escherichia coli ST3204 strain coproducing NDM-16 and MCR-1.

A plasmid-mediated colistin resistance gene, mcr-1, has been reported worldwide and has caused concern regarding a major therapeutic challenge. Alarmingly, mcr-1 has spread into clinical carbapenem-resistant Enterobacteriaceae isolates, resulting in extensively drug-resistant and even pan drug-resistant isolates that can cause untreatable infections. In this study, we report isolation of an extensively drug-resistant Escherichia coli strain EC1188 that coproduces NDM-16 and MCR-1 from a urine sample taken from a patient with craniocerebral injury.E. coli strain EC1188 was identified and subjected to genotyping, susceptibility testing and conjugation experiments. The genetic locations of blaNDM-16 and mcr-1 were established with southern blot hybridization. The complete genome sequence of this strain was obtained and the genetic characteristics of the mcr-1- and blaNDM-16-harboring plasmids were analyzed. In addition, comparative genetic analyses of mcr-1 and blaNDM-16 with closely related plasmids were also carried out.Whole-genome sequencing revealed that strain EC1188 possess various resistance genes and virulence genes. S1-pulsed-field gel electrophoresis and southern blot suggested that the blaNDM-16 and mcr-1 genes were located on an ~65 kb plasmid and an ~80 kb plasmid, respectively. Moreover, the two genes could successfully transfer their resistance phenotype to E. coli strain C600. Sequence analysis showed that these two plasmids possessed high sequence similarity to previously reported blaNDM-5-harboring and mcr-1-harboring plasmids in China.To the best of our knowledge, this is the first report to isolate an E. coli strain that coproduces NDM-16 and MCR-1. In addition, we characterized the blaNDM-16-harboring plasmid for the first time. Our study further emphasizes that the co-occurrence of the two prevalent transferrable resistance plasmids in a single isolate is highly significant because infections caused by MCR-1-producing carbapenem-resistant Enterobacteriaceae isolates are increasing each year. It is imperative to perform active surveillance to prevent further dissemination of MCR-1-producing CRE isolates.

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September 22, 2019

Discovery of multi-drug resistant, MCR-1 and ESBL-coproducing ST117 Escherichia coli from diseased chickens in Northeast China

An endemic multi-drug resistant ST117 E. coli isolate coproducing MCR-1 and 3 ESBL loci was, for the first time, detected from diseased chicken, Liaoning Province, in Northeast China, from 2011 to 2012. Whole-genome sequencing revealed 5 unique plasmids, namely pHXH-1, pHXH-2, pHXH-3, pHXH-4 and pHXH-5). Among them, pHXH1 and pHXH4 encode ESBL, and pHXH-5 mediates MCR-1 colistin resistance. The results indicate that the potentially-national dissemination of MCR-1-positive pathogens with pan-drug resistance proceeds via food chains.

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September 22, 2019

Comparative genomics reveal a flagellar system, a type VI secretion system and plant growth-promoting gene clusters unique to the endophytic bacterium Kosakonia radicincitans.

The recent worldwide discovery of plant growth-promoting (PGP) Kosakonia radicincitans in a large variety of crop plants suggests that this species confers significant influence on plants, both in terms of yield increase and product quality improvement. We provide a comparative genome analysis which helps to unravel the genetic basis for K. radicincitans’ motility, competitiveness and plant growth-promoting capacities. We discovered that K. radicincitans carries multiple copies of complex gene clusters, among them two flagellar systems and three type VI secretion systems (T6SSs). We speculate that host invasion may be facilitated by different flagella, and bacterial competitor suppression by effector proteins ejected via T6SSs. We found a large plasmid in K. radicincitans DSM 16656T, the species type strain, that confers the potential to exploit plant-derived carbon sources. We propose that multiple copies of complex gene clusters in K. radicincitans are metabolically expensive but provide competitive advantage over other bacterial strains in nutrient-rich environments. The comparison of the DSM 16656T genome to genomes of other genera of enteric plant growth-promoting bacteria (PGPB) exhibits traits unique to DSM 16656T and K. radicincitans, respectively, and traits shared between genera. We used the output of the in silico analysis for predicting the purpose of genomic features unique to K. radicincitans and performed microarray, PhyloChip, and microscopical analyses to gain deeper insight into the interaction of DSM 16656T, plants and associated microbiota. The comparative genome analysis will facilitate the future search for promising candidates of PGPB for sustainable crop production.

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September 22, 2019

Comparison of highly and weakly virulent Dickeya solani strains, with a view on the pangenome and panregulon of this species.

Bacteria belonging to the genera Dickeya and Pectobacterium are responsible for significant economic losses in a wide variety of crops and ornamentals. During last years, increasing losses in potato production have been attributed to the appearance of Dickeya solani. The D. solani strains investigated so far share genetic homogeneity, although different virulence levels were observed among strains of various origins. The purpose of this study was to investigate the genetic traits possibly related to the diverse virulence levels by means of comparative genomics. First, we developed a new genome assembly pipeline which allowed us to complete the D. solani genomes. Four de novo sequenced and ten publicly available genomes were used to identify the structure of the D. solani pangenome, in which 74.8 and 25.2% of genes were grouped into the core and dispensable genome, respectively. For D. solani panregulon analysis, we performed a binding site prediction for four transcription factors, namely CRP, KdgR, PecS and Fur, to detect the regulons of these virulence regulators. Most of the D. solani potential virulence factors were predicted to belong to the accessory regulons of CRP, KdgR, and PecS. Thus, some differences in gene expression could exist between D. solani strains. The comparison between a highly and a low virulent strain, IFB0099 and IFB0223, respectively, disclosed only small differences between their genomes but significant differences in the production of virulence factors like pectinases, cellulases and proteases, and in their mobility. The D. solani strains also diverge in the number and size of prophages present in their genomes. Another relevant difference is the disruption of the adhesin gene fhaB2 in the highly virulent strain. Strain IFB0223, which has a complete adhesin gene, is less mobile and less aggressive than IFB0099. This suggests that in this case, mobility rather than adherence is needed in order to trigger disease symptoms. This study highlights the utility of comparative genomics in predicting D. solani traits involved in the aggressiveness of this emerging plant pathogen.

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September 22, 2019

Genomic analysis for heavy metal resistance in S. maltophilia

Stenotrophomonas maltophilia is highly resistant to heavy metals, but the genetic knowledge of metal resistance in S. maltophilia is poorly understood. In this study, the genome of S. maltophilia Pho isolated from the contaminated soil near a metalwork factory was sequenced using PacBio RS II. Its genome is composed of a single chromosome with a GC content of 66.4% and 4434 protein-encoding genes. Comparative analysis revealed high syntney between S. maltophilia Pho and the model strain, S. maltophilia K279a. Then, the type and number of mechanisms of heavy metal uptake were analyzed firstly. Results showed that 7 unspecific ion transporter genes and 13 specific ion transporter genes, most of which were involved in iron transport. But the sulfate permeases belonging to the family of SulT/CysP that can uptake chromate and the high affinity ZnuABC/SitABCD were absent. Secondly, the putative genes controlling metal efflux were analyzed. Results showed that this bacterium encoded 5 CDFs, 1 copper exporting ATPase and 4 RND systems, including 2 CzcABC efflux pumps. Moreover, the putative metal transformation genes including arsenate and mercury detoxification genes were also identified. This study may provide useful information on the metal resistance mechanisms of S. maltophilia.

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September 22, 2019

Changes in the genetic requirements for microbial interactions with increasing community complexity.

Microbial community structure and function rely on complex interactions whose underlying molecular mechanisms are poorly understood. To investigate these interactions in a simple microbiome, we introduced E. coli into an experimental community based on a cheese rind and identified the differences in E. coli’s genetic requirements for growth in interactive and non-interactive contexts using Random Barcode Transposon Sequencing (RB-TnSeq) and RNASeq. Genetic requirements varied among pairwise growth conditions and between pairwise and community conditions. Our analysis points to mechanisms by which growth conditions change as a result of increasing community complexity and suggests that growth within a community relies on a combination of pairwise and higher-order interactions. Our work provides a framework for using the model organism E. coli as a readout to investigate microbial interactions regardless of the genetic tractability of members of the studied ecosystem.© 2018, Morin et al.

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September 22, 2019

Involvement of Burkholderiaceae and sulfurous volatiles in disease-suppressive soils.

Disease-suppressive soils are ecosystems in which plants suffer less from root infections due to the activities of specific microbial consortia. The characteristics of soils suppressive to specific fungal root pathogens are comparable to those of adaptive immunity in animals, as reported by Raaijmakers and Mazzola (Science 352:1392-3, 2016), but the mechanisms and microbial species involved in the soil suppressiveness are largely unknown. Previous taxonomic and metatranscriptome analyses of a soil suppressive to the fungal root pathogen Rhizoctonia solani revealed that members of the Burkholderiaceae family were more abundant and more active in suppressive than in non-suppressive soils. Here, isolation, phylogeny, and soil bioassays revealed a significant disease-suppressive activity for representative isolates of Burkholderia pyrrocinia, Paraburkholderia caledonica, P. graminis, P. hospita, and P. terricola. In vitro antifungal activity was only observed for P. graminis. Comparative genomics and metabolite profiling further showed that the antifungal activity of P. graminis PHS1 was associated with the production of sulfurous volatile compounds encoded by genes not found in the other four genera. Site-directed mutagenesis of two of these genes, encoding a dimethyl sulfoxide reductase and a cysteine desulfurase, resulted in a loss of antifungal activity both in vitro and in situ. These results indicate that specific members of the Burkholderiaceae family contribute to soil suppressiveness via the production of sulfurous volatile compounds.

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September 22, 2019

Genomic analysis of multidrug-resistant Escherichia coli ST58 causing urosepsis.

Sequence type 58 (ST58) phylogroup B1 Escherichia coli have been isolated from a wide variety of mammalian and avian hosts but are not noted for their ability to cause serious disease in humans or animals. Here we determined the genome sequences of two multidrug-resistant E. coli ST58 strains from urine and blood of one patient using a combination of Illumina and Single Molecule, Real-Time (SMRT) sequencing. Both ST58 strains were clonal and were characterised as serotype O8:H25, phylogroup B1 and carried a complex resistance locus/loci (CRL) that featured an atypical class 1 integron with a dfrA5 (trimethoprim resistance) gene cassette followed by only 24 bp of the 3′-CS. CRL that carry this particular integron have been described previously in E. coli from cattle, pigs and humans in Australia. The integron abuts a copy of Tn6029, an IS26-flanked composite transposon encoding blaTEM, sul2 and strAB genes that confer resistance to ampicillin, sulfathiazole and streptomycin, respectively. The CRL resides within a novel Tn2610-like hybrid Tn1721/Tn21 transposon on an IncF, ColV plasmid (pSDJ2009-52F) of 138 553 bp that encodes virulence associated genes implicated in life-threatening extraintestinal pathogenic E. coli (ExPEC) infections. Notably, pSDJ2009-52F shares high sequence identity with pSF-088-1, a plasmid reported in an E. coli ST95 strain from a patient with blood sepsis from a hospital in San Francisco. These data suggest that extraintestinal infections caused by E. coli carrying ColV-like plasmids, irrespective of their phylogroup or ST, may pose a potential threat to human health, particularly to the elderly and immunocompromised. Copyright © 2018. Published by Elsevier B.V.

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September 22, 2019

Characterization of a novel SXT/R391 Integrative and Conjugative Element carrying cfr, blaCTX-M-65, fosA3 and aac(6′)-Ib-cr in Proteus mirabilis.

A novel 139,487-bp SXT/R391 integrative and conjugative element, ICEPmiChnBCP11, was characterized in Proteus mirabilis of swine origin in China. ICEPmiChnBCP11 harbors 20 different antimicrobial resistance genes, including the clinically important rRNA methyltransferase gene cfr, the extended-spectrum ß-lactamase gene blaCTX-M-65, fosfomycin resistance gene fosA3, and fluoroquinolone resistance gene aac(6′)-Ib-cr An ISPpu12-mediated composite transposon containing various resistance genes and 10 copies of IS26 is inserted in hot spot 4. ICEPmiChnBCP11 was successfully transferred to Escherichia coli. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

A model for the evolution of prokaryotic DNA restriction-modification systems based upon the structural malleability of Type I restriction-modification enzymes.

Restriction Modification (RM) systems prevent the invasion of foreign genetic material into bacterial cells by restriction and protect the host’s genetic material by methylation. They are therefore important in maintaining the integrity of the host genome. RM systems are currently classified into four types (I to IV) on the basis of differences in composition, target recognition, cofactors and the manner in which they cleave DNA. Comparing the structures of the different types, similarities can be observed suggesting an evolutionary link between these different types. This work describes the ‘deconstruction’ of a large Type I RM enzyme into forms structurally similar to smaller Type II RM enzymes in an effort to elucidate the pathway taken by Nature to form these different RM enzymes. Based upon the ability to engineer new enzymes from the Type I ‘scaffold’, an evolutionary pathway and the evolutionary pressures required to move along the pathway from Type I RM systems to Type II RM systems are proposed. Experiments to test the evolutionary model are discussed.

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September 22, 2019

Evolutionary history of human Plasmodium vivax revealed by genome-wide analyses of related ape parasites.

Wild-living African apes are endemically infected with parasites that are closely related to human Plasmodium vivax, a leading cause of malaria outside Africa. This finding suggests that the origin of P. vivax was in Africa, even though the parasite is now rare in humans there. To elucidate the emergence of human P. vivax and its relationship to the ape parasites, we analyzed genome sequence data of P. vivax strains infecting six chimpanzees and one gorilla from Cameroon, Gabon, and Côte d’Ivoire. We found that ape and human parasites share nearly identical core genomes, differing by only 2% of coding sequences. However, compared with the ape parasites, human strains of P. vivax exhibit about 10-fold less diversity and have a relative excess of nonsynonymous nucleotide polymorphisms, with site-frequency spectra suggesting they are subject to greatly relaxed purifying selection. These data suggest that human P. vivax has undergone an extreme bottleneck, followed by rapid population expansion. Investigating potential host-specificity determinants, we found that ape P. vivax parasites encode intact orthologs of three reticulocyte-binding protein genes (rbp2d, rbp2e, and rbp3), which are pseudogenes in all human P. vivax strains. However, binding studies of recombinant RBP2e and RBP3 proteins to human, chimpanzee, and gorilla erythrocytes revealed no evidence of host-specific barriers to red blood cell invasion. These data suggest that, from an ancient stock of P. vivax parasites capable of infecting both humans and apes, a severely bottlenecked lineage emerged out of Africa and underwent rapid population growth as it spread globally. Copyright © 2018 the Author(s). Published by PNAS.

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September 22, 2019

Genome analyses of the microalga Picochlorum provide insights into the evolution of thermotolerance in the green lineage.

While the molecular events involved in cell responses to heat stress have been extensively studied, our understanding of the genetic basis of basal thermotolerance, and particularly its evolution within the green lineage, remains limited. Here, we present the 13.3-Mb haploid genome and transcriptomes of a halotolerant and thermotolerant unicellular green alga, Picochlorum costavermella (Trebouxiophyceae) to investigate the evolution of the genomic basis of thermotolerance. Differential gene expression at high and standard temperatures revealed that more of the gene families containing up-regulated genes at high temperature were recently evolved, and less originated at the ancestor of green plants. Inversely, there was an excess of ancient gene families containing transcriptionally repressed genes. Interestingly, there is a striking overlap between the thermotolerance and halotolerance transcriptional rewiring, as more than one-third of the gene families up-regulated at 35?°C were also up-regulated under variable salt concentrations in Picochlorum SE3. Moreover, phylogenetic analysis of the 9,304 protein coding genes revealed 26 genes of horizontally transferred origin in P. costavermella, of which five were differentially expressed at higher temperature. Altogether, these results provide new insights about how the genomic basis of adaptation to halo- and thermotolerance evolved in the green lineage.

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September 22, 2019

The structure of a conserved telomeric region associated with variant antigen loci in the blood parasite Trypanosoma congolense

African trypanosomiasis is a vector-borne disease of humans and livestock caused by African trypanosomes (Trypanosoma spp.). Survival in the vertebrate bloodstream depends on antigenic variation of Variant Surface Glycoproteins (VSGs) coating the parasite surface. In T. brucei, a model for antigenic variation, monoallelic VSG expression originates from dedicated VSG expression sites (VES). Trypanosoma brucei VES have a conserved structure consisting of a telomeric VSG locus downstream of unique, repeat sequences, and an independent promoter. Additional protein-coding sequences, known as “Expression Site Associated Genes (ESAGs)”, are also often present and are implicated in diverse, bloodstream-stage functions. Trypanosoma congolense is a related veterinary pathogen, also displaying VSG-mediated antigenic variation. A T. congolense VES has not been described, making it unclear if regulation of VSG expression is conserved between species. Here, we describe a conserved telomeric region associated with VSG loci from long-read DNA sequencing of two T. congolense strains, which consists of a distal repeat, conserved noncoding elements and other genes besides the VSG; although these are not orthologous to T. brucei ESAGs. Most conserved telomeric regions are associated with accessory minichromosomes, but the same structure may also be associated with megabase chromosomes. We propose that this region represents the T. congolense VES, and through comparison with T. brucei, we discuss the parallel evolution of antigenic switching mechanisms, and unique adaptation of the T. brucei VES for developmental regulation of bloodstream-stage genes. Hence, we provide a basis for understanding antigenic switching in T. congolense and the origins of the African trypanosome VES.

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September 22, 2019

Genome alterations associated with improved transformation efficiency in Lactobacillus reuteri.

Lactic acid bacteria (LAB) are one of the microorganisms of choice for the development of protein delivery systems for therapeutic purposes. Although there are numerous tools to facilitate genome engineering of lactobacilli; transformation efficiency still limits the ability to engineer their genomes. While genetically manipulating Lactobacillus reuteri ATCC PTA 6475 (LR 6475), we noticed that after an initial transformation, several LR 6475 strains significantly improved their ability to take up plasmid DNA via electroporation. Our goal was to understand the molecular basis for how these strains acquired the ability to increase transformation efficiency.Strains generated after transformation of plasmids pJP067 and pJP042 increased their ability to transform plasmid DNA about one million fold for pJP067, 100-fold for pSIP411 and tenfold for pNZ8048. Upon sequencing of the whole genome from these strains, we identified several genomic mutations and rearrangements, with all strains containing mutations in the transformation related gene A (trgA). To evaluate the role of trgA in transformation of DNA, we generated a trgA null that improved the transformation efficiency of LR 6475 to transform pSIP411 and pJP067 by at least 100-fold, demonstrating that trgA significantly impairs the ability of LR 6475 to take-up plasmid DNA. We also identified genomic rearrangements located in and around two prophages inserted in the LR 6475 genome that included deletions, insertions and an inversion of 336 Kb. A second group of rearrangements was observed in a Type I restriction modification system, in which the specificity subunits underwent several rearrangements in the target recognition domain. Despite the magnitude of these rearrangements in the prophage genomes and restriction modification systems, none of these genomic changes impacted transformation efficiency to the level induced by trgA.Our findings demonstrate how genetic manipulation of LR 6475 with plasmid DNA leads to genomic changes that improve their ability to transform plasmid DNA; highlighting trgA as the primary driver of this phenotype. Additionally, this study also underlines the importance of characterizing genetic changes that take place after genome engineering of strains for therapeutic purposes.

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