Engineering conjugative CRISPR-Cas9 systems for the targeted control of enteric pathogens and antibiotic resistance.

Pathogenic Escherichia coli and Salmonella enterica pose serious public health threats due to their ability to cause severe gastroenteritis and life-threatening sequela, particularly in young children. Moreover, the emergence and dissemination of antibiotic resistance in these bacteria have complicated control of infections. Alternative strategies that effectively target these enteric pathogens and negate or reduce the need of antibiotics are urgently needed. Such an alternative is the CRISPR-Cas9 system because it can generate sequence-specific lethal double stranded DNA breaks. In this study, two self-transmissible broad host range conjugative plasmids, pRK24 and pBP136, were engineered to deliver multiplexed CRSIPR-Cas9 systems that specifically target Enterohemorrhagic and Enteropathogenic strains of E. coli (EHEC and EPEC), S. enterica, and blaCMY-2 antibiotic resistance plasmids. Using in vitro mating assays, we show that the conjugative delivery of pRK24-CRISPR-Cas9 carrying guide RNAs to the EPEC/EHEC eae (intimin) gene can selectively kill enterohemorrhagic E. coli O157 eae+ cells (3 log kill at 6 h) but does not kill the isogenic Δeae mutant (P<0.001). Similar results were also obtained with a pBP136 derivative, pTF16, carrying multiplexed guide RNAs targeting E. coli eae and the S. enterica ssaN gene coding for the type III secretion ATPase. Another pBP136 derivative, TF18, carries guide RNAs targeting S. enterica ssaN and the antibiotic resistance gene, blaCMY-2, carried on the multi-drug resistant pAR06302. Introduction of pTF18 into bacteria harboring pAR06302 showed plasmids were cured at an efficiency of 53% (P<0.05). Using a murine neonate EPEC infection model, pTF16 was delivered by a murine derived E. coli strain to EPEC infected mice and showed significant reductions of intestinal EPEC (P<0.05). These results suggest that establishing conjugative CRISPR-Cas9 antimicrobials in the intestinal microbiome may provide protection from enteric pathogens and reduce antibiotic resistance without disrupting the normal microbiota.

Initial responses of the trap-crop, Solanum sisymbriifolium, to Globodera pallida invasions.

Many researchers today are looking for mechanisms underlying plant defenses against nematodes by identifying differentially expressed genes in domesticated hosts. In order to provide a different perspective, we analyzed the root transcriptome of an undomesticated non-host species, Solanum sisymbriifolium Lamark (SSI) before and after Globodera pallida infection. Utilizing RNAseq analyses, we identified changes in the expression of 277 transcripts. Many of these genes were not annotated; however, the annotated set included peroxidases, reactive oxygen species-producing proteins, and regulators of cell death. Importantly, 60% of the nematode-responsive genes did not respond to physical damage to root tissues, or to exogenous treatments with either salicylic acid or methyl jasmonate. Based on this, we speculate that the majority of changes in SSI gene expression were promoted by either nematode effectors, pathogen-associated molecular patterns (PAMPs), or by exposure to untested endogenous signaling molecules such as ethylene, or by exposure to multiple stimuli. This study incorporates our findings into a model that accounts for part of this plant's unusual resistance to nematodes.

Assessment of an Organ-Specific de Novo Transcriptome of the Nematode Trap-Crop, Solanum sisymbriifolium.

Solanum sisymbriifolium, also known as "Litchi Tomato" or "Sticky Nightshade," is an undomesticated and poorly researched plant related to potato and tomato. Unlike the latter species, S. sisymbriifolium induces eggs of the cyst nematode, Globodera pallida, to hatch and migrate into its roots, but then arrests further nematode maturation. In order to provide researchers with a partial blueprint of its genetic make-up so that the mechanism of this response might be identified, we used single molecule real time (SMRT) sequencing to compile a high quality de novo transcriptome of 41,189 unigenes drawn from individually sequenced bud, root, stem, and leaf RNA populations. Functional annotation and BUSCO analysis showed that this transcriptome was surprisingly complete, even though it represented genes expressed at a single time point. By sequencing the 4 organ libraries separately, we found we could get a reliable snapshot of transcript distributions in each organ. A divergent site analysis of the merged transcriptome indicated that this species might have undergone a recent genome duplication and re-diploidization. Further analysis indicated that the plant then retained a disproportionate number of genes associated with photosynthesis and amino acid metabolism in comparison to genes with characteristics of R-proteins or involved in secondary metabolism. The former processes may have given S. sisymbriifolium a bigger competitive advantage than the latter did.

Assessment of Globodera pallida RNA Extracted from Solanum Roots.

The introduction of high-throughput sequencing technologies has made transcriptome analyses of plant-pathogen interactions almost routine. Nevertheless, it is still challenging to obtain RNA from populations made up of two species. An RNA extraction method that worked well on free-living Caenorhabditis elegans failed when applied to isolated Globodera pallida J2 larva. Furthermore, alternative protocols that extracted RNA from free-living J2 larva produced less satisfactory results once the animals entered their hosts' roots. We have compared several extraction procedures to ascertain whether a single protocol was capable of recovering high-quality, high-molecular-weight RNA from newly hatched J2 larva as well as from larva embedded in roots of both potatoes (Solanum tuberosum L. cv. Desiree) and a very distantly related species, Solanum sisymbriifolium. Although it was possible to recover large amounts of RNA from J2 larvae using Proteinase K treatments, this protocol failed to yield high-quality nematode RNA from infected roots. By comparison, mechanical disruption procedures yielded lower amounts of RNA from infected roots, but what was recovered was of higher quality. We conclude that different extraction protocols need to be developed to sample mixed populations of organisms.

Physiological levels of glucose induce membrane vesicle secretion and affect the lipid and protein composition of Yersinia pestis cell surfaces.

Yersinia pestis grown with physiologic glucose increased cell autoaggregation and deposition of extracellular material, including membrane vesicles. Membranes were characterized, and glucose had significant effects on protein, lipid, and carbohydrate profiles. These effects were independent of temperature and the biofilm-related locus pgm and were not observed in Yersinia pseudotuberculosis.

Autophagy proteins play cytoprotective and cytocidal roles in leucine starvation-induced cell death in Saccharomyces cerevisiae.

Autophagy is essential for prolonging yeast survival during nutrient deprivation; however, this report shows that some autophagy proteins may also be accelerating population death in those conditions. While leucine starvation caused YCA1-mediated apoptosis characterized by increased annexin V staining, nitrogen deprivation triggered necrotic death characterized by increased propidium iodide uptake. Although a Δatg8 strain died faster than its parental strain during nitrogen starvation, this mutant died slower than its parent during leucine starvation. Conversely, a Δatg11 strain died slower than its parent during nitrogen starvation, but faster during leucine starvation. Curiously, although GFP-Atg8 complemented the Δatg8 mutation, this protein made ATG8 cells more sensitive to nitrogen starvation, and less sensitive to leucine starvation. These results were difficult to explain if autophagy only extended life but could be an indication that a second form of autophagy could concurrently facilitate either apoptotic or necrotic cell death.

Identification of autophagy genes participating in zinc-induced necrotic cell death in Saccharomyces cerevisiae.

Eukaryotes use a common set of genes to perform two mechanistically similar autophagic processes. Bulk autophagy harvests proteins nonselectively and reuses their constitutents when nutrients are scarce. In contrast, different forms of selective autophagy target protein aggregates or damaged organelles that threaten to interfere with growth. Yeast uses one form of selective autophagy, called cytoplasm-to-vacuole targeting (Cvt), to engulf two vacuolar enzymes in Cvt vesicles ("CVT-somes") within which they are transported to vacuoles for maturation. While both are dispensable normally, bulk and selective autophagy help sustain life under stressful conditions. Consistent with this view, knocking out several genes participating in Cvt and specialized autophagic pathways heightened the sensitivity of Saccharomyces cerevisiae to inhibitory levels of Zn(2+). The loss of other autophagic genes, and genes responsible for apoptotic cell death, had no such effect. Unexpectedly, the loss of members of a third set of autophagy genes heightened cellular resistance to zinc as if they encoded proteins that actively contributed to zinc-induced cell death. Further studies showed that both sensitive and resistant strains accumulated similar amounts of H2O2 during zinc treatments, but that more sensitive strains showed signs of necrosis sooner. Although zinc lethality depended on autophagic proteins, studies with several reporter genes failed to reveal increased autophagic activity. In fact, microscopy analysis indicated that Zn(2+) partially inhibited fusion of Cvt vesicles with vacuoles. Further studies into how the loss of autophagic processes suppressed necrosis in yeast might reveal whether a similar process could occur in plants and animals.

Structural, functional, and evolutionary analysis of moeZ, a gene encoding an enzyme required for the synthesis of the Pseudomonas metabolite, pyridine-2,6-bis(thiocarboxylic acid).

BACKGROUND: Pyridine-2,6-bis(thiocarboxylic acid) (pdtc) is a small secreted metabolite that has a high affinity for transition metals, increases iron uptake efficiency by 20% in Pseudomonas stutzeri, has the ability to reduce both soluble and mineral forms of iron, and has antimicrobial activity towards several species of bacteria. Six GenBank sequences code for proteins similar in structure to MoeZ, a P. stutzeri protein necessary for the synthesis of pdtc. RESULTS: Analysis of sequences similar to P. stutzeri MoeZ revealed that it is a member of a superfamily consisting of related but structurally distinct proteins that are members of pathways involved in the transfer of sulfur-containing moieties to metabolites. Members of this family of enzymes are referred to here as MoeB, MoeBR, MoeZ, and MoeZdR. MoeB, the molybdopterin synthase activating enzyme in the molybdopterin cofactor biosynthesis pathway, is the most characterized protein from this family. Remarkably, lengths of greater than 73% nucleic acid homology ranging from 35 to 486 bp exist between Pseudomonas stutzeri moeZ and genomic sequences found in some Mycobacterium, Mesorhizobium, Pseudomonas, Streptomyces, and cyanobacteria species. CONCLUSIONS: The phylogenetic relationship among moeZ sequences suggests that P. stutzeri may have acquired moeZ through lateral gene transfer from a donor more closely related to mycobacteria and cyanobacteria than to proteobacteria. The importance of this relationship lies in the fact that pdtc, the product of the P. stutzeri pathway that includes moeZ, has an impressive set of capabilities, some of which could make it a potent pathogenicity factor.

Screening and characterization of aldehyde dehydrogenase gene from Halomonas salina strain AS11.

A population survey was made of moderately halophilic bacteria in prawn pond sediment in the Songkla region of Thailand. Twenty-two isolated halophilic bacteria capable of growing on modified ATCC culture medium 1270 for halobacterium were then assayed for aldehyde dehydrogenase (ALDH) activity which might be involved in the metabolism of xenobiotic compounds. One isolate, designated AS11, was selected based on its high amount of ALDH activity. This organism can grow at sodium chloride concentrations ranging from 2.5 to 25%, although optimum growth occurs at 5% NaCl. Phenotypic and phylogenetic studies indicated that AS11 was an isolate of Halomonas salina. The aldh gene coding for this enzyme was then cloned. The open reading frame of the aldh gene was 1521-bp long and coded for a protein of 506 amino acid residues with a calculated molecular mass of 55 kDa. The aldh gene product proved to be 76% identical to the NAD-dependent acetaldehyde dehydrogenase gene from Pseudomonase aeruginosa.