Transkingdom Genetic Transfer from Escherichia coli to Saccharomyces cerevisiae as a Simple Gene Introduction Tool

Transkingdom conjugation (TKC) permits transfer of DNA from bacteria to eukaryotic cells using a bacterial conjugal transfer system. However, it is not clear whether the process of DNA acceptance in a recipient eukaryote is homologous to the process of conjugation between bacteria. TKC transfer requires mobilizable shuttle vectors that are capable of conjugal transfer and replication in the donor and recipient strains. Here, we developed TKC vectors derived from plasmids belonging to the IncP and IncQ groups. We also investigated forms of transfer of these vectors from Escherichia coli into Saccharomyces cerevisiae to develop TKC as a simple gene introduction method. Both types of vectors were transferred precisely, conserving the origin of transfer (oriT) sequences, but IncP-based vectors appeared to be more efficient than an IncQ-based vector. Interestingly, unlike in agrobacterial T-DNA (transfer DNA) transfer, the efficiency of TKC transfer was similar between a wild-type yeast strain and DNA repair mutants defective in homologous recombination (rad51Δ and rad52Δ) or nonhomologous end joining (rad50Δ, yku70Δ, and lig4Δ). Lastly, a shuttle vector with two repeats of IncP-type oriT (oriT^P) sequences flanking a marker gene was constructed. TKC transfer of this vector resulted in precise excision of both the oriT^P loci as well as the marker gene, albeit at a low frequency of 17% of all transconjugants. This feature would be attractive in biotechnological applications of TKC. Taken together, these results strongly suggest that in contrast to agrobacterial T-DNA transfer, the circularization of vector single-stranded DNA occurs either before or after transfer but requires a factor(s) from the donor. TKC is a simple method of gene transfer with possible applications in yeast genetics and biotechnology.     

Novel plasmid-based genetic tools for the study of promoters and terminators in Streptococcus pneumoniae and Enterococcus faecalis

Promoter-probe and terminator-probe plasmid vectors make possible to rapidly examine whether particular sequences function as promoter or terminator signals in various genetic backgrounds and under diverse environmental stimuli. At present, such plasmid-based genetic tools are very scarce in the Gram-positive pathogenic bacteria Streptococcus pneumoniae and Enterococcus faecalis. Hence, we developed novel promoter-probe and terminator-probe vectors based on the Streptococcus agalactiae pMV158 plasmid, which replicates autonomously in numerous Gram-positive bacteria. As reporter gene, a gfp allele encoding a variant of the green fluorescent protein was used. These genetic tools were shown to be suitable to assess the activity of promoters and terminators (both homologous and heterologous) in S. pneumoniae and E. faecalis. In addition, the promoter-probe vector was shown to be a valuable tool for the analysis of regulated promoters in vivo, such as the promoter of the pneumococcal fuculose kinase gene. These new plasmid vectors will be very useful for the experimental verification of predicted promoter and terminator sequences, as well as for the construction of new inducible-expression vectors. Given the promiscuity exhibited by the pMV158 replicon, these vectors could be used in a variety of Gram-positive bacteria.     

Induction of Hydrolytic Enzymes in Brassica campestris in Response to Pathovars of Xanthomonas campestris

Inoculation of mature leaves of turnip (Brassica campestris) with the incompatible Xanthomonas campestris pv vitians resulted in the induction of β-1,3-glucanase and chitinase/lysozyme (CHL) activity. No increase in the basal activity of β-1,3-glucanase was observed after inoculation of leaves with heat- or rifampicin-killed X. c. vitians, Escherichia coli, or sterile water. Inoculation with the compatible X. campestris pv campestris resulted in a slower induction of glucanase than that seen with X. c. vitians. In contrast, all bacteria caused an induction of CHL activity. One major β-1,3-glucanase (molecular mass 36.5 kilodaltons, isoelectric point [pl] ~8.5) was purified from both inoculated and untreated leaves by ion-exchange chromatography. The enzyme degraded laminarin by an endo-glycolytic mechanism. Two major CHL isozymes (CHL 1 and CHL 2, molecular mass 30 kilodaltons and pl 9.4 and 10.2, respectively) were purified from X. c. vitians inoculated leaves by affinity chromatography on a chitin column followed by ion-exchange chromatography. Both enzymes degraded chitin by an endo-glycolytic mechanism although the ratio of lysozyme to chitinase specific activities for CHL 1 and CHL2 were different. The induction of CHL 1 was associated with the hypersensitive reaction caused by X. c. vitians whereas all other treatments induced largely CHL 2.     

The N-Glycan Cluster from Xanthomonas campestris pv. campestris: A TOOLBOX FOR SEQUENTIAL PLANT N-GLYCAN PROCESSING*

N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three mannoses, one fucose, and one xylose (N₂M₃FX) as a substrate. The removal of the α-1,3-mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the β-xylosidase NixI (GH3), which is involved in the cleavage of the β-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.     

pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates “clean-gene” facilities

A new plasmid series has been created for Agrobacterium-mediated plant transformation. The pBECKS2000 series of binary vectors exploits the Cre/loxP site-specific recombinase system to facilitate the construction of complex T-DNA vectors. The new plasmids enable the rapid generation of T-DNA vectors in which multiple genes are linked, without relying on the availability of purpose-built cassette systems or demanding complex, and therefore inefficient, ligation reactions. The vectors incorporate facilities for the removal of transformation markers from transgenic plants, while still permitting simple in vitro manipulations of the T-DNA vectors. A `shuttle' or intermediate plasmid approach has been employed. This permits independent ligation strategies to be used for two gene sets. The intermediate plasmid sequence is incorporated into the binary vector through a plasmid co-integration reaction which is mediated by the Cre/loxP site-specific recombinase system. This reaction is carried out within Agrobacterium cells. Recombinant clones, carrying the co-integrative binary plasmid form, are selected directly using the antibiotic resistance marker carried on the intermediate plasmid. This strategy facilitates production of co-integrative T-DNA binary vector forms which are appropriate for either (1) transfer to and integration within the plant genome of target and marker genes as a single T-DNA unit; (2) transfer and integration of target and marker genes as a single T-DNA unit but with a Cre/loxP facility for site-specific excision of marker genes from the plant genome; or (3) co-transfer of target and marker genes as two independent T-DNAs within a single-strain Agrobacterium system, providing the potential for segregational loss of marker genes.     

Applications for green fluorescent protein (GFP) in the study of hostpathogen interactions

The green fluorescent protein (GFP) from Aequorea victoria is a novel fluorescent marker that has potential use in the study of bacterial pathogenicity. To explore some of the potential applications of GFP to the study of host-parasite interactions, we constructed two GFP expression vectors suitable for different facultative intracellular bacterial pathogens. The first expression vector was tested in the enteric pathogens, Salmonella typhimurium and Yersinia pseudotuberculosis, and the second vector tested in Mycobacterium marinum (Mm). Both expression vectors were found to be stable and to direct high levels of GFP synthesis. Standard epifluorescence microscopy was used to detect all three bacterial pathogenic species during the early and late stages of infection of live mammalian cells. Mm expressing gfp was also visualized in infected animal tissues, gfp expression did not adversely affect bacterial survival, nor did it compromise entry into mammalian cells or their survival within macrophages. In addition, all three gfp-expressing bacterial pathogens could be detected and sorted in a flow cytometer, either alone or in association with epithelial cells or macrophages. Therefore, GFP not only provides a convenient tool to image pathogenic bacteria, but allows the quantitative measurement of bacterial association with mammalian cells.     

Improvement of the Coprinopsis cinerea molecular toolkit using new construct design and additional marker genes

This paper describes the optimisation of an existing basidiomycete molecular toolkit through the development of new versatile vectors. These vectors enable the straightforward and rapid construction of gene expression and silencing cassettes by allowing the easy exchange of promoters, coding regions and terminator elements. The constructs contain multiple cloning sites (MCS) allowing any gene to be inserted using a range of restriction sites, with the option of a 5′ integral intron for efficient gene expression. We describe the testing of these vectors through marker gene expression in Coprinopsis cinerea. This work also extends the range of marker genes available for use in C. cinerea with the first report of DsRed and monomeric red fluorescent protein (mRFP) expression in C. cinerea and further demonstrates the requirement for an intron in the expression cassette for some marker genes. However, analysis of transformants containing either β-glucuronidase (GUS) or luciferase (LUC) genes, with and without an intron revealed no detectable marker gene expression. The inclusion of an intron does therefore not guarantee expression and other genetic factors may be involved.     

Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants

Genetic transformation is one of the most widely used technique in crop improvement. However, most of the binary vectors used in this technique, especially cloning based, contain antibiotic genes as selection marker that raise serious consumer and environmental concerns; moreover, they could be transferred to non-target hosts with deleterious effects. Therefore, the goal of this study was reconstruction of the widely used pBI121 binary vector by substituting the harmful antibiotic selection marker gene with a less-harmful selection marker, Basta (herbicide resistance gene). The generated vectors were designated as pBI121NB and pBI121CB, in which Basta gene was expressed under the control of Nos or CaMV 35S promoter, respectively. The successful integration of the new inserts into both the vectors was confirmed by PCR, restriction digestion and sequencing. Both these vectors were used in transforming Arabidopsis, Egyptian wheat and barley varieties using LBA4404 and GV3101 Agrobacterium strains. The surfactant Tween-20 resulted in an efficient transformation and the number of Arabidopsis transformants was about 6–9 %. Soaked seeds of wheat and barley were transformed with Agrobacterium to introduce the bacteria to the growing shoot apices. The percentage of transgenic lines was around 16–17 and 14–15 % for wheat and barley, respectively. The quantitative studies presented in this work showed that both LBA4404 and GV3101 strains were suitable for transforming Egyptian wheat and barley.     

Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 × 10⁶ and 7.1 × 10⁶ transformants/μg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.     

Genetic and Proteomic Analyses of a Xanthomonas campestris pv. campestris purC Mutant Deficient in Purine Biosynthesis and Virulence

Bacterial proliferation in hosts requires activation of a number of housekeeping pathways, including purine de novo biosynthesis. Although inactivation of purine biosynthesis genes can attenuate virulence, it is unclear which biochemical or virulence factors are associated with the purine biosynthesis pathway in vivo. We report that inactivation of purC, a gene encoding phosphoribosylaminoimidazole-succinocarboxamide synthase, caused complete loss of virulence in Xanthomonas campestris pv. cam- pestris, the causal agent of black rot disease of cruciferous plants. The purC mutant was a purine auxotroph; it could not grow on minimal medium, whereas addition of purine derivatives, such as hypoxanthine or adenine plus guanine, restored growth of the mutant. The purC mutation also significantly enhanced the production of an unknown purine synthesis associated pigment and extracellular polysaccharides by the bacterium. In addition, comparative proteomic analyses of bacteria grown on rich and minimal media revealed that the purC mutation affected the expression levels of diverse proteins involved in purine and pyrimidine synthesis, carbon and energy metabolisms, iron uptake, proteolysis, protein secretion, and signal transduction. These results provided clues to understanding the contributions of purine synthesis to bacterial virulence and interactions with host immune systems.     

Yeast vectors for integration at the HO locus

We have constructed new yeast vectors for targeted integration of desired sequences at the Saccharomyces cerevisiae HO locus. Insertion at HO has been shown to have no effect on yeast growth, and thus these integrations should be neutral. One vector contains the KanMX selectable marker, and integrants can be selected by resistance to G418. The other vector contains the hisG-URA3-hisG cassette, and integrants can be selected by uracil prototrophy. Subsequent growth on 5-FOA permits identification of colonies where recombination between the hisG tandem repeats has led to loss of the URA3 marker and return to uracil auxotrophy. We also describe several new bacterial polylinker vectors derived from pUC21 (ampicillin resistance) and pUK21 (kanamycin resistance).     

Versatile plasmid-based expression systems for Gram-negative bacteria—General essentials exemplified with the bacterium Ralstonia eutropha H16

The Gram-negative bacterium Escherichia coli is currently the most efficient and widely used prokaryotic host for recombinant protein and metabolite production. However, due to some limitations and to various interesting features of other Gram-negative bacteria efficient vector systems applicable to a broad range are desired. Basic building blocks for plasmid-based vectors include besides the need for a suitable selection marker in the first line a proper replication and maintenance system. In addition to these basic requirements, further elements are needed for Gram-negative bacteria beyond E. coli, such as Pseudomonas pudita, Ralstonia eutropha, Burkholderia glumae or Acinetobacter sp.. Established building blocks have to be adapted and new building blocks providing the desired functions need to be identified and exploited. This minireview addresses so far described and used genetic elements for broad host range replication, efficient plasmid maintenance, and conjugative plasmid transfer as well as expression elements and protein secretion signals. The industrially important bacterium R. eutropha H16 was chosen as a model organism to provide specific data on the effectivity and utility of building blocks based on such genetic elements.     

Saccharomyces cerevisiae-Based Molecular Tool Kit for Manipulation of Genes from Gram-Negative Bacteria

A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.     

Reproducible and Quantitative Model of Infection of Dermacentor variabilis with the Live Vaccine Strain of Francisella tularensis

Pathogen life cycles in mammalian hosts have been studied extensively, but studies with arthropod vectors represent considerable challenges. In part this is due to the difficulty of delivering a reproducible dose of bacteria to follow arthropod-associated replication. We have established reproducible techniques to introduce known numbers of Francisella tularensis strain LVS from mice into Dermacentor variabilis nymphs. Using this model infection system, we performed dose-response infection experiments and followed bacterial replication through the molt to adults and at later time points. During development to adults, bacteria replicate to high numbers and can be found associated with the gut tissues, salivary glands, and hemolymph of adult ticks. Further, we can transmit a mutant of LVS (LVS ΔpurMCD) that cannot replicate in macrophages in vitro or in mice to nymphs. Our data show that the LVS ΔpurMCD mutant cannot be transstadially transmitted from nymphs to adult ticks. We then show that a plasmid-complemented strain of this mutant is recoverable in adult ticks and necessary for bacterial replication during the molt. In a mixed-infection assay (ΔpurMCD mutant versus ΔpurMCD complement), 98% of the recovered bacteria retained the plasmid marker. These data suggest that the ΔpurMCD mutation cannot be rescued by the presence a complemented strain in a mixed infection. Importantly, our infection model provides a platform to test specific mutants for their replication in ticks, perform competition studies, and use other genetic techniques to identify F. tularensis genes that are expressed or required in this unique environment.     

Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3''-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.     

Identification and Regulation of the N-Acetylglucosamine Utilization Pathway of the Plant Pathogenic Bacterium Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris, the causal agent of black rot disease of brassicas, is known for its ability to catabolize a wide range of plant compounds. This ability is correlated with the presence of specific carbohydrate utilization loci containing TonB-dependent transporters (CUT loci) devoted to scavenging specific carbohydrates. In this study, we demonstrate that there is an X. campestris pv. campestris CUT system involved in the import and catabolism of N-acetylglucosamine (GlcNAc). Expression of genes belonging to this GlcNAc CUT system is under the control of GlcNAc via the LacI family NagR and GntR family NagQ regulators. Analysis of the NagR and NagQ regulons confirmed that GlcNAc utilization involves NagA and NagB-II enzymes responsible for the conversion of GlcNAc-6-phosphate to fructose-6-phosphate. Mutants with mutations in the corresponding genes are sensitive to GlcNAc, as previously reported for Escherichia coli. This GlcNAc sensitivity and analysis of the NagQ and NagR regulons were used to dissect the X. campestris pv. campestris GlcNAc utilization pathway. This analysis revealed specific features, including the fact that uptake of GlcNAc through the inner membrane occurs via a major facilitator superfamily transporter and the fact that this amino sugar is phosphorylated by two proteins belonging to the glucokinase family, NagK-IIA and NagK-IIB. However, NagK-IIA seems to play a more important role in GlcNAc utilization than NagK-IIB under our experimental conditions. The X. campestris pv. campestris GlcNAc NagR regulon includes four genes encoding TonB-dependent active transporters (TBDTs). However, the results of transport experiments suggest that GlcNAc passively diffuses through the bacterial envelope, an observation that calls into question whether GlcNAc is a natural substrate for these TBDTs and consequently is the source of GlcNAc for this nonchitinolytic plant-associated bacterium.Xanthomonas campestris pv. campestris, the causal agent of black rot disease of brassicas, produces extracellular plant cell wall-degrading enzymes which contribute to its pathogenicity by facilitating its spread through plant tissues and give the bacterium access to a ready source of nutrients via the carbohydrate utilization loci containing TonB-dependent transporters (CUT loci) (7, 16, 35). The CUT loci are characterized by the presence of genes encoding regulators, degradative enzymes, inner membrane transporters, and outer membrane TonB-dependent transporters (TBDTs), which have been identified as active carbohydrate transporters (7, 33, 44). However, recently, an example of passive diffusion through a TBDT in Caulobacter crescentus was described (17). X. campestris pv. campestris has 72 TBDTs and belongs to a class of bacteria in which TBDTs are overrepresented (7). Our previous study suggested that there are several CUT loci or systems in this bacterium (7).N-Acetylglucosamine (GlcNAc) is an amino sugar that is used for the synthesis of cell surface structures in bacteria and plays an important role in supplying carbon and energy by entering the glycolytic pathway after it is converted into fructose-6-phosphate (fructose-6P) (1, 9). In a recent comparative study of bacterial GlcNAc utilization pathways and regulatory networks, Yang and coworkers identified conserved and distinct features of the GlcNAc utilization pathway in proteobacteria (48). The expression of X. campestris pv. campestris GlcNAc-specific genes was proposed to be controlled by NagR and NagQ regulators belonging to the LacI and GntR families, respectively. In X. campestris pv. campestris strain ATCC 33913, one predicted binding motif specific for NagQ (designated the NagQ box) consists of two imperfect repeats of the TGGTATT sequence separated by 4 bp and is located upstream of the nagQ gene (XCC3414) (Fig. ​(Fig.1A)1A) (48). This gene is part of the nag cluster and is followed by genes encoding the major facilitator superfamily (MFS) inner membrane transporter NagP (XCC3413), the regulator NagR (XCC3412), the GlcN-6P deaminase NagB-II (XCC3411), and the GlcNAc-6P deacetylase NagA (XCC3410) (Fig. ​(Fig.1A).1A). NagR boxes contain the palindromic sequence AATGACARCGYTGTCATT (bold type indicates less highly conserved nucleotides) and are upstream of genes encoding two glucokinase-like NagK-II proteins (XCC2886 [nagK-IIA] and XCC2943 [nagK-IIB]), as well as 5 genes encoding TBDTs (XCC0531, XCC2887, XCC3045, XCC3408, and XCC2944 located downstream of XCC2943) (Fig. ​(Fig.1A).1A). All of the X. campestris pv. campestris genes located downstream of NagR or NagQ boxes were proposed to belong to a GlcNAc utilization pathway involved in uptake of GlcNAc through the bacterial envelope and subsequent phosphorylation, deacetylation, and deamination, which finally leads to the common metabolic intermediate fructose-6-phosphate (Fig. ​(Fig.1B)1B) (48). It was recently demonstrated that in C. crescentus the TBDT CC0446 gene, which is clustered with other nag genes, is responsible for the uptake of GlcNAc (17). The presence of TBDTs in the GlcNAc regulon, which has been observed in Alteromonadales and Xanthomonadales (48), suggests that genes belonging to the GlcNAc utilization pathway define a new CUT system.Open in a separate windowFIG. 1.X. campestris pv. campestris N-acetylglucosamine (GlcNAc) utilization pathway. (A) Organization of genes in the proposed GlcNAc utilization pathway. NagR boxes are indicated by filled circles, and the NagQ box is indicated by an open circle. (B) GlcNAc is proposed to be transported through the outer membrane by TBDTs and then transported across the inner membrane by the MFS transporter NagP. GlcNAc would then be phosphorylated by nagK-II-encoded enzymes. Subsequent metabolism via the nagA-encoded (GlcNAc-6P deacetylase) and nagB-II-encoded (GlcN-6P deaminase) enzymes results in fructose 6-phosphate (Fru-6P) (48). MFS, major facilitator superfamily; PP, periplasm; TBDT, TonB-dependent transporter.Here we describe characterization of the X. campestris pv. campestris GlcNAc utilization pathway and regulatory network, which involves at least the repressors NagR and NagQ. TBDTs are associated with this pathway, confirming the presence of a GlcNAc CUT system in X. campestris pv. campestris. In this bacterium, GlcNAc entry and catabolism imply that novel families containing a GlcNAc inner membrane transporter and GlcNAc kinases are involved.     

Comparative analysis of the gut microbiota of sand fly vectors of zoonotic visceral leishmaniasis (ZVL) in Iran; host-environment interplay shapes diversity

The development of Leishmania parasites within sand fly vectors occurs entirely in the insect gut lumen, in the presence of symbiotic and commensal bacteria. The impacts of host species and environment on the gut microbiome are currently poorly understood. We employed MiSeq sequencing of the V3-16S rRNA gene amplicons to characterize and compare the gut microbiota of field-collected populations of Phlebotomus kandelakii, P. perfiliewi, P. alexandri, and P. major, the primary or secondary vectors of zoonotic visceral leishmaniasis (ZVL) in three distinct regions of Iran where ZVL is endemic. In total, 160,550 quality-filtered reads of the V3 region yielded a total of 72 operational taxonomic units (OTUs), belonging to 23 phyla, 47 classes, 91 orders, 131 families, and 335 genera. More than 50% of the bacteria identified were Proteobacteria, followed by Firmicutes (22%), Deinococcus-Thermus (9%), Actinobacteria (6%), and Bacteroidetes (5%). The core microbiome was dominated by eight genera: Acinetobacter, Streptococcus, Enterococcus, Staphylococcus, Bacillus, Propionibacterium, Kocuria, and Corynebacterium. Wolbachia were found in P. alexandri and P. perfiliewi, while Asaia sp. was reported in P. perfiliewi. Substantial variations in the gut bacterial composition were found between geographically distinct populations of the same sand fly species, as well as between different species at the same location, suggesting that sand fly gut microbiota is shaped by both the host species and geographical location. Phlebotomus kandelakii and P. perfiliewi in the northwest, and P. alexandri in the south, the major ZVL vectors, harbor the highest bacterial diversity, suggesting a possible relationship between microbiome diversity and the capacity for parasite transmission. In addition, large numbers of gram-positive human or animal pathogens were found, suggesting that sand fly vectors of ZVL could pose a potential additional threat to livestock and humans in the region studied. The presence of Bacillus subtilis, Enterobacter cloacae, and Asaia sp suggests that these bacteria could be promising candidates for a paratransgenesis approach to the fight against Leishmaniasis.     

thyA as a Selection Marker in Construction of Food-Grade Host-Vector and Integration Systems for Streptococcus thermophilus

We constructed food-grade host-vector and integration systems for Streptococcus thermophilus by using a thymidylate synthase gene (thyA) as the selection marker. Two thyA genes, thyA_St and thyA_Lb, were cloned from S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, respectively. Thymidine-requiring mutants of S. thermophilus were obtained after successive cultures in the presence of trimethoprim, and one of them, TM1-1, was used as the host. Food-grade vectors were constructed by using either thyA_St or thyA_Lb as the selection marker. Transformants of TM1-1 created by using these vectors were selected for thymidine autotrophy as efficiently as for erythromycin resistance. By using the host-vector system developed in this way, a foreign amylase gene (amyA) was expressed in TM1-1 and was also integrated into the chromosome by use of a temperature-sensitive integration vector constructed with thyA_Lb as the selection marker via a double-crossover event. The results obtained show that thyA is an efficient and safe selection marker for S. thermophilus that is suitable for food applications.     

Development of New Modular Genetic Tools for Engineering the Halophilic Archaeon Halobacterium salinarum

Our ability to genetically manipulate living organisms is usually constrained by the efficiency of the genetic tools available for the system of interest. In this report, we present the design, construction and characterization of a set of four new modular vectors, the pHsal series, for engineering Halobacterium salinarum, a model halophilic archaeon widely used in systems biology studies. The pHsal shuttle vectors are organized in four modules: (i) the E. coli’s specific part, containing a ColE1 origin of replication and an ampicillin resistance marker, (ii) the resistance marker and (iii) the replication origin, which are specific to H. salinarum and (iv) the cargo, which will carry a sequence of interest cloned in a multiple cloning site, flanked by universal M13 primers. Each module was constructed using only minimal functional elements that were sequence edited to eliminate redundant restriction sites useful for cloning. This optimization process allowed the construction of vectors with reduced sizes compared to currently available platforms and expanded multiple cloning sites. Additionally, the strong constitutive promoter of the fer2 gene was sequence optimized and incorporated into the platform to allow high-level expression of heterologous genes in H. salinarum. The system also includes a new minimal suicide vector for the generation of knockouts and/or the incorporation of chromosomal tags, as well as a vector for promoter probing using a GFP gene as reporter. This new set of optimized vectors should strongly facilitate the engineering of H. salinarum and similar strategies could be implemented for other archaea.