Functional binding dynamics relevant to the evolution of zoonotic spillovers in endemic and emergent Betacoronavirus strains.

Comparative functional analysis of the dynamic interactions between various Betacoronavirus mutant strains and broadly utilized target proteins such as ACE2 and CD26, is crucial for a more complete understanding of zoonotic spillovers of viruses that cause diseases such as COVID-19. Here, we employ machine learning to replicated sets of nanosecond scale GPU accelerated molecular dynamics simulations to statistically compare and classify atom motions of these target proteins in both the presence and absence of different endemic and emergent strains of the viral receptor binding domain (RBD) of the S spike glycoprotein. A multi-agent classifier successfully identified functional binding dynamics that are evolutionarily conserved from bat CoV-HKU4 to human endemic/emergent strains. Conserved dynamics regions of ACE2 involve both the N-terminal helices, as well as a region of more transient dynamics encompassing residues K353, Q325 and a novel motif AAQPFLL 386-92 that appears to coordinate their dynamic interactions with the viral RBD at N501. We also demonstrate that the functional evolution of Betacoronavirus zoonotic spillovers involving ACE2 interaction dynamics are likely pre-adapted from two precise and stable binding sites involving the viral bat progenitor strain's interaction with CD26 at SAMLI 291-5 and SS 333-334. Our analyses further indicate that the human endemic strains hCoV-HKU1 and hCoV-OC43 have evolved more stable N-terminal helix interactions through enhancement of an interfacing loop region on the viral RBD, whereas the highly transmissible SARS-CoV-2 variants (B.1.1.7, B.1.351 and P.1) have evolved more stable viral binding via more focused interactions between the viral N501 and ACE2 K353 alone.Communicated by Ramaswamy H. Sarma.

Function and evolution of B-Raf loop dynamics relevant to cancer recurrence under drug inhibition.

Oncogenic mutations in the kinase domain of the B-Raf protein have long been associated with cancers involving the MAPK pathway. One constitutive MAPK activating mutation in B-Raf, the V600E (valine to glutamate) replacement occurring adjacent to a site of threonine phosphorylation (T599) occurs in many types of cancer, and in a large percentage of certain cancers, such as melanoma. Because ATP binding activity and the V600E mutation are both known to alter the physical behavior of the activation loop in the B-Raf ATP binding domain, this system is especially amenable to comparative analyses of molecular dynamics simulations modeling various genetic and drug class variants. Here, we employ machine learning enabled identification of functionally conserved protein dynamics to compare how the binding interactions of four B-Raf inhibitors impact the functional loop dynamics controlling ATP activation. We demonstrate that drug development targeting B-Raf has progressively moved towards ATP competitive inhibitors that demonstrate less tendency to mimic the functionally conserved dynamic changes associated with ATP activation and leading to the side effect of hyperactivation (i.e. inducing MAPK activation in non-tumorous cells in the absence of secondary mutation). We compare the functional dynamic impacts of V600E and other sensitizing and drug resistance causing mutations in the regulatory loops of B-Raf, confirming sites of low mutational tolerance in these regions. Lastly, we investigate V600E sensitivity of B-Raf loop dynamics in an evolutionary context, demonstrating that while sensitivity has an ancient origin with primitive eukaryotes, it was also secondarily increased during early jawed vertebrate evolution.Communicated by Ramaswamy H. Sarma.

Expression of a Shiga-Like Toxin during Plastic Colonization by Two Multidrug-Resistant Bacteria, Aeromonas hydrophila RIT668 and Citrobacter freundii RIT669, Isolated from Endangered Turtles (Clemmys guttata).

Aeromonas hydrophila RIT668 and Citrobacter freundii RIT669 were isolated from endangered spotted turtles (Clemmys guttata). Whole-genome sequencing, annotation and phylogenetic analyses of the genomes revealed that the closest relative of RIT668 is A. hydrophila ATCC 7966 and Citrobacter portucalensis A60 for RIT669. Resistome analysis showed that A. hydrophila and C. freundii harbor six and 19 different antibiotic resistance genes, respectively. Both bacteria colonize polyethylene and polypropylene, which are common plastics, found in the environment and are used to fabricate medical devices. The expression of six biofilm-related genes-biofilm peroxide resistance protein (bsmA), biofilm formation regulatory protein subunit R (bssR), biofilm formation regulatory protein subunit S (bssS), biofilm formation regulator (hmsP), toxin-antitoxin biofilm protein (tabA) and transcriptional activator of curli operon (csgD)-and two virulence factors-Vi antigen-related gene (viaB) and Shiga-like toxin (slt-II)-was investigated by RT-PCR. A. hydrophila displayed a >2-fold increase in slt-II expression in cells adhering to both polymers, C. freundii adhering on polyethylene displayed a >2-fold, and on polypropylene a >6-fold upregulation of slt-II. Thus, the two new isolates are potential pathogens owing to their drug resistance, surface colonization and upregulation of a slt-II-type diarrheal toxin on polymer surfaces.

Insight Into the Microbial Co-occurrence and Diversity of 73 Grapevine (Vitis vinifera) Crown Galls Collected Across the Northern Hemisphere.

Crown gall (CG) is a globally distributed and economically important disease of grapevine and other important crop plants. The causal agent of CG is Agrobacterium or Allorhizobium strains that harbor a tumor-inducing plasmid (pTi). The microbial community within the CG tumor has not been widely elucidated and it is not known if certain members of this microbial community promote or inhibit CG. This study investigated the microbiotas of grapevine CG tumor tissues from seven infected vineyards located in Hungary, Japan, Tunisia, and the United States. Heavy co-amplification of grapevine chloroplast and mitochondrial ribosomal RNA genes was observed with the widely used Illumina V3-V4 16S rRNA gene primers, requiring the design of a new reverse primer to enrich for bacterial 16S rRNA from CG tumors. The operational taxonomic unit (OTU) clustering approach is not suitable for CG microbiota analysis as it collapsed several ecologically distinct Agrobacterium species into a single OTU due to low interspecies genetic divergence. The CG microbial community assemblages were significantly different across sampling sites (ANOSIM global R = 0.63, p-value = 0.001) with evidence of site-specific differentially abundant ASVs. The presence of Allorhizobium vitis in the CG microbiota is almost always accompanied by Xanthomonas and Novosphingobium, the latter may promote the spread of pTi plasmid by way of acyl-homoserine lactone signal production, whereas the former may take advantage of the presence of substrates associated with plant cell wall growth and repair. The technical and biological insights gained from this study will contribute to the understanding of complex interaction between the grapevine and its microbial community and may facilitate better management of CG disease in the future.

The crystal structure of dihydrodipicolinate reductase from the human-pathogenic bacterium Bartonella henselae strain Houston-1 at 2.3†Šresolution.

In bacteria, the second committed step in the diaminopimelate/lysine anabolic pathways is catalyzed by the enzyme dihydrodipicolinate reductase (DapB). DapB catalyzes the reduction of dihydrodipicolinate to yield tetrahydrodipicolinate. Here, the cloning, expression, purification, crystallization and X-ray diffraction analysis of DapB from the human-pathogenic bacterium Bartonella henselae, the causative bacterium of cat-scratch disease, are reported. Protein crystals were grown in conditions consisting of 5%(w/v) PEG 4000, 200 mM sodium acetate, 100 mM sodium citrate tribasic pH 5.5 and were shown to diffract to ∼2.3 Šresolution. They belonged to space group P4322, with unit-cell parameters a = 109.38, b = 109.38, c = 176.95 Å. Rr.i.m. was 0.11, Rwork was 0.177 and Rfree was 0.208. The three-dimensional structural features of the enzymes show that DapB from B. henselae is a tetramer consisting of four identical polypeptides. In addition, the substrate NADP+ was found to be bound to one monomer, which resulted in a closed conformational change in the N-terminal domain.

Genome sequence of Novosphingobium sp. strain Rr 2-17, a nopaline crown gall-associated bacterium isolated from Vitis vinifera L. grapevine.

Novosphingobium sp. strain Rr 2-17 is an N-acyl homoserine lactone (AHL)-producing bacterium isolated from the crown gall tumor of a grapevine. To our knowledge, this is the first draft genome announcement of a plant-associated strain from the genus Novosphingobium.

Identification of an rsh gene from a Novosphingobium sp. necessary for quorum-sensing signal accumulation.

The stringent response is a mechanism by which bacteria adapt to environmental stresses and nutritional deficiencies through the synthesis and hydrolysis of (p)ppGpp by RelA/SpoT enzymes. Alphaproteobacteria and plants contain a single Rsh enzyme (named for RelA/SpoT homolog) that is bifunctional. Here we report the identification of a new species of bacteria belonging to the genus Novosphingobium and characterization of an rsh mutation in this plant tumor-associated isolate. Isolate Rr 2-17, from a grapevine crown gall tumor, is a member of the Novosphingobium genus that produces the N-acyl-homoserine lactone (AHL) quorum-sensing (QS) signals. A Tn5 mutant, Hx 699, deficient in AHL production was found to have an insertion in an rsh gene. The Rsh protein showed significant percent sequence identity to Rsh proteins of alphaproteobacteria. The Novosphingobium sp. rsh gene (rsh(Nsp)) complemented the multiple amino acid requirements of the Escherichia coli relA spoT double mutant by restoring the growth on selection media. Besides QS signal production, the rsh mutation also affects soluble polysaccharide production and cell aggregation. Genetic complementation of the Hx 699 mutant with the rsh(Nsp) gene restored these phenotypes. This is the first discovery of a functional rsh gene in a member of the Novosphingobium genus.

Localization of members of the gamma-glutamyl transpeptidase family identifies sites of glutathione and glutathione S-conjugate hydrolysis.

gamma-Glutamyl transpeptidases (GGTs) are essential for hydrolysis of the tripeptide glutathione (gamma-glutamate-cysteine-glycine) and glutathione S-conjugates since they are the only enzymes known to cleave the amide bond linking the gamma-carboxylate of glutamate to cysteine. In Arabidopsis thaliana, four GGT genes have been identified based on homology with animal GGTs. They are designated GGT1 (At4g39640), GGT2 (At4g39650), GGT3 (At1g69820), and GGT4 (At4g29210). By analyzing the expression of each GGT in plants containing GGT:beta-glucuronidase fusions, the temporal and spatial pattern of degradation of glutathione and its metabolites was established, revealing appreciable overlap among GGTs. GGT2 exhibited narrow temporal and spatial expression primarily in immature trichomes, developing seeds, and pollen. GGT1 and GGT3 were coexpressed in most organs/tissues. Their expression was highest at sites of rapid growth including the rosette apex, floral stem apex, and seeds and might pinpoint locations where glutathione is delivered to sink tissues to supplement high demand for cysteine. In mature tissues, they were expressed only in vascular tissue. Knockout mutants of GGT2 and GGT4 showed no phenotype. The rosettes of GGT1 knockouts showed premature senescence after flowering. Knockouts of GGT3 showed reduced number of siliques and reduced seed yield. Knockouts were used to localize and assign catalytic activity to each GGT. In the standard GGT assay with gamma-glutamyl p-nitroanilide as substrate, GGT1 accounted for 80% to 99% of the activity in all tissues except seeds where GGT2 was 50% of the activity. Protoplasting experiments indicated that both GGT1 and GGT2 are localized extracellularly but have different physical or chemical associations.

Methionine and threonine synthesis are limited by homoserine availability and not the activity of homoserine kinase in Arabidopsis thaliana.

Homoserine kinase (HSK) produces O-phospho-l-homoserine (HserP) used by cystathionine gamma-synthase (CGS) for Met synthesis and threonine synthase (TS) for Thr synthesis. The effects of overexpressing Arabidopsis thaliana HSK, CGS, and Escherichia coli TS (eTS), each controlled by the 35S promoter, were compared. The results indicate that in Arabidopsis Hser supply is the major factor limiting the synthesis of HserP, Met and Thr. HSK is not limiting and CGS or TS control the partitioning of HserP. HSK overexpression had no effect on the level of soluble HserP, Met or Thr, however, when treated with Hser these plants produced far more HserP than wild type. Met and Thr also accumulated markedly after Hser treatment but the increase was similar in HSK overexpressing and wild-type plants. CGS overexpression was previously shown to increase Met content, but had no effect on Thr. After Hser treatment Met accumulation increased in CGS-overexpressing plants compared with wild type, whereas HserP declined and Thr was unaffected. Arabidopsis responded differentially to eTS expression depending on the level of the enzyme. At the highest eTS level the Thr content was not increased, but the phenotype was negatively affected and the T1 plants died before reproducing. Comparatively low eTS did not affect phenotype or Thr/Met level, however after Hser treatment HserP and Met accumulation were reduced compared with wild type and Thr was increased slightly. At intermediate eTS activity seedling growth was retarded unless Met was supplied and CGS expression was induced, indicating that eTS limited HserP availability for Met synthesis.

Biosynthesis of lysine in plants: evidence for a variant of the known bacterial pathways.

With the aim of elucidating how plants synthesize lysine, extracts prepared from corn, tobacco, Chlamydomonas and soybean were tested and found to lack detectable amounts of N-alpha-acyl-L,L-diaminopimelate deacylase or N-succinyl-alpha-amino-epsilon-ketopimelate-glutamate aminotransaminase, two key enzymes in the central part of the bacterial pathway for lysine biosynthesis. Corn extracts missing two key enzymes still carried out the overall synthesis of lysine when provided with dihydrodipicolinate. An analysis of available plant DNA sequences was performed to test the veracity of the negative biochemical findings. Orthologs of dihydrodipicolinate reductase and diaminopimelate epimerase (enzymes on each side of the central pathway) were readily found in the Arabidopsis thaliana genome. Orthologs of the known enzymes needed to convert tetrahydrodipicolinate to diaminopimelic acid (DAP) were not detected in Arabidopsis or in the plant DNA sequence databases. The biochemical and reinforcing bioinformatics results provide evidence that plants may use a novel variant of the bacterial pathways for lysine biosynthesis.