Whole-genome sequencing of four culturable endophytic bacteria from German hardneck garlic cloves (Allium sativum L.).

We present the whole-genome sequence of four bacterial endophytes associated with German hardneck garlic cloves (Allium sativum L.). Among them, Agrobacterium fabrum and Pantoea agglomerans are associated with plant protection, while Rahnella perminowiae and Stenotrophomonas lactitubi are pathogens. These data will facilitate the identification of genes to improve garlic.

Nucleic acid-based small molecules as targeted transcription therapeutics for immunoregulation.

Transcription therapy is an emerging approach that centers on identifying the factors associated with the malfunctioning gene transcription machinery that causes diseases and controlling them with designer agents. Until now, the primary research focus in therapeutic gene modulation has been on small-molecule drugs that target epigenetic enzymes and critical signaling pathways. However, nucleic acid-based small molecules have gained popularity in recent years for their amenability to be pre-designed and realize operative control over the dynamic transcription machinery that governs how the immune system responds to diseases. Pyrrole-imidazole polyamides (PIPs) are well-established DNA-based small-molecule gene regulators that overcome the limitations of their conventional counterparts owing to their sequence-targeted specificity, versatile regulatory efficiency, and biocompatibility. Here, we emphasize the rational design of PIPs, their functional mechanisms, and their potential as targeted transcription therapeutics for disease treatment by regulating the immune response. Furthermore, we also discuss the challenges and foresight of this approach in personalized immunotherapy in precision medicine.

Bat-associated microbes: Opportunities and perils, an overview.

The potential biotechnological uses of bat-associated bacteria are discussed briefly, indicating avenues for biotechnological applications of bat-associated microbes. The uniqueness of bats in terms of their lifestyle, genomes and molecular immunology may predispose bats to act as disease reservoirs. Molecular phylogenetic analysis has shown several instances of bats harbouring the ancestral lineages of bacterial (Bartonella), protozoal (Plasmodium, Trypanosoma cruzi) and viral (SARS-CoV2) pathogens infecting humans. Along with the transmission of viruses from bats, we also discuss the potential roles of bat-associated bacteria, fungi, and protozoan parasites in emerging diseases. Current evidence suggests that environmental changes and interactions between wildlife, livestock, and humans contribute to the spill-over of infectious agents from bats to other hosts. Domestic animals including livestock may act as intermediate amplifying hosts for bat-origin pathogens to transmit to humans. An increasing number of studies investigating bat pathogen diversity and infection dynamics have been published. However, whether or how these infectious agents are transmitted both within bat populations and to other hosts, including humans, often remains unknown. Metagenomic approaches are uncovering the dynamics and distribution of potential pathogens in bat microbiomes, which might improve the understanding of disease emergence and transmission. Here, we summarize the current knowledge on bat zoonoses of public health concern and flag the gaps in the knowledge to enable further research and allocation of resources for tackling future outbreaks.

Endophytic bacteria associated with wild-type banana seed (Musa balbisiana): whole genome sequencing.

We present the whole-genome sequences of five endophytic bacteria isolated from Musa balbisiana seeds. These strains represent five different genera: Bacillus, Brachybacterium, Enterobacter, Enterococcus, and Pantoea. Among these, three genera (Bacillus, Pantoea, and Enterobacter) were previously recognized for their antagonistic effects against Fusarium wilt, a highly destructive disease that affects banana plants.

Chromatographic isolation of potentially novel antibiotic compounds produced by Yimella sp. RIT 621.

OBJECTIVE: Antibiotic resistant infections have become a global health crisis causing 1.2 million deaths worldwide in 2019 [1]. In a previous study, we identified a bacterium from a rare genus, Yimella, and found in an initial antibiotic screening that they produce broad-spectrum bactericidal compounds [2]. Herein, we focus on the characterization of these potential novel antimicrobial compounds produced by Yimella sp. RIT 621. RESULTS: We used solid-phase extraction and C18 reverse-phase chromatography to isolate the antibiotic-active compounds found in organic extracts from liquid cultures of Yimella sp. RIT 621. We tracked the antimicrobial activity by testing the extracts in disc diffusion inhibitory assays and observed its increase after each purification stage.

Polystyrene Degradation by Exiguobacterium sp. RIT 594: Preliminary Evidence for a Pathway Containing an Atypical Oxygenase.

The widespread use of plastics has led to their increasing presence in the environment and subsequent pollution. Some microorganisms degrade plastics in natural ecosystems and the associated metabolic pathways can be studied to understand the degradation mechanisms. Polystyrene (PS) is one of the more recalcitrant plastic polymers that is degraded by only a few bacteria. Exiguobacterium is a genus of Gram-positive poly-extremophilic bacteria known to degrade PS, thus being of biotechnological interest, but its biochemical mechanisms of degradation have not yet been elucidated. Based solely on genome annotation, we initially proposed PS degradation by Exiguobacterium sp. RIT 594 via depolymerization and epoxidation catalyzed by a ring epoxidase. However, Fourier transform infrared (FTIR) spectroscopy analysis revealed an increase of carboxyl and hydroxyl groups with biodegradation, as well as of unconjugated C-C double bonds, both consistent with dearomatization of the styrene ring. This excludes any aerobic pathways involving side chain epoxidation and/or hydroxylation. Subsequent experiments confirmed that molecular oxygen is critical to PS degradation by RIT 594 because degradation ceased under oxygen-deprived conditions. Our studies suggest that styrene breakdown by this bacterium occurs via the sequential action of two enzymes encoded in the genome: an orphan aromatic ring-cleaving dioxygenase and a hydrolase.

Isolation, Whole-Genome Sequencing, and Annotation of Three Unclassified Antibiotic-Producing Bacteria, Enterobacter sp. Strain RIT 637, Pseudomonas sp. Strain RIT 778, and Deinococcus sp. Strain RIT 780.

We report the isolation, whole-genome sequencing, and annotation of Enterobacter sp. strain RIT 637, Pseudomonas sp. strain RIT 778, and Deinococcus sp. strain RIT 780. Disk diffusion assays using spent medium demonstrated that all bacteria produced bactericidal compounds against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923.

Exiguobacterium sp. is endowed with antibiotic properties against Gram positive and negative bacteria.

OBJECTIVE: In order to isolate and identify bacteria that produce potentially novel bactericidal/bacteriostatic compounds, two ponds on the campus of the Rochester Institute of Technology (RIT) were targeted as part of a bioprospecting effort. RESULTS: One of the unique isolates, RIT 452 was identified as Exiguobacterium sp. and subjected to whole-genome sequencing. The genome was assembled and in silico analysis was performed to predict the secondary metabolite gene clusters, which suggested the potential of Exiguobacterium RIT452 for producing antibiotic compounds. Extracts of spent growth media of RIT452 were active in disc diffusion assays performed against four reference strains, two Gram-negative (E. coli ATCC 25922 and P. aeruginosa ATCC 27853) and two Gram-positive (B. subtilis BGSC 168 and S. aureus ATCC 25923). Differential extraction and liquid chromatography was used to fractionate the extracts. Efforts to identify and elucidate the structure of the active compound(s) are still ongoing.

Amino acid-derived defense metabolites from plants: A potential source to facilitate novel antimicrobial development.

For millennia, humanity has relied on plants for its medicines, and modern pharmacology continues to reexamine and mine plant metabolites for novel compounds and to guide improvements in biological activity, bioavailability, and chemical stability. The critical problem of antibiotic resistance and increasing exposure to viral and parasitic diseases has spurred renewed interest into drug treatments for infectious diseases. In this context, an urgent revival of natural product discovery is globally underway with special attention directed toward the numerous and chemically diverse plant defensive compounds such as phytoalexins and phytoanticipins that combat herbivores, microbial pathogens, or competing plants. Moreover, advancements in "omics," chemistry, and heterologous expression systems have facilitated the purification and characterization of plant metabolites and the identification of possible therapeutic targets. In this review, we describe several important amino acid-derived classes of plant defensive compounds, including antimicrobial peptides (e.g., defensins, thionins, and knottins), alkaloids, nonproteogenic amino acids, and phenylpropanoids as potential drug leads, examining their mechanisms of action, therapeutic targets, and structure-function relationships. Given their potent antibacterial, antifungal, antiparasitic, and antiviral properties, which can be superior to existing drugs, phytoalexins and phytoanticipins are an excellent resource to facilitate the rational design and development of antimicrobial drugs.

Antileishmanial Drug Development: A Review of Modern Molecular Chemical Tools and Research Strategies.

Leishmaniasis, a complex disease caused by at least 20 species of unicellular parasites of the genus Leishmania, disproportionately affects impoverished regions of about 90 tropical and sub-tropical countries. Currently available antileishmanial therapies, particularly for visceral leishmaniasis, are severely limited, with treatment outcome depending on many factors, including the immune status of the patient, comorbidities, malnutrition, and socio-economic conditions in the patient's geographic location. There is an urgent need for new therapeutics, particularly new effective oral drugs, for visceral leishmaniasis. Despite the availability of the Leishmania genome sequence information and significant research into the biology of the parasites, antileishmanial drug development is hampered by the lack of knowledge about druggable targets in the parasite and difficulties in identifying the molecular targets of compounds that show activity. In this context, we analyzed recent progress in antileishmanial drug development programs, which take advantage of different powerful approaches, such as high-throughput screening of compound libraries, recent developments in genetic methods for assessing essentiality of parasite genes, and chemical, genetic, and proteomics-based target discovery and target validation methods.

Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669-Potential Zoonotic Pathogens Isolated from Spotted Turtles.

Antimicrobial resistance (AMR) is one of the biggest challenges of the 21st century, and biofilm formation enables bacteria to resist antibiotic at much higher concentrations than planktonic cells. Earlier, we showed that the Gram-negative Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669 (closely related to C. freundii NBRC 12681) from infected spotted turtles (Clemmys guttata), formed biofilms and upregulated toxin expression on plastic surfaces, and were predicted to possess multiple antibiotic resistance genes. Here, we show that they each resist several antibiotics in the planktonic phase, but were susceptible to neomycin, and high concentrations of tetracycline and cotrimoxazole. The susceptibility of their biofilms to neomycin and cotrimoxazole was tested using the Calgary device. For A. hydrophila, the minimum inhibitory concentration (MIC) = 500-1000, and the minimum biofilm eradication concentration (MBEC) > 1000 µg/mL, using cotrimoxazole, and MIC = 32.3-62.5, and MBEC > 1000 µg/mL, using neomycin. For C. freundii MIC = 7.8-15.6, and, MBEC > 1000 µg/mL, using cotrimoxazole, and MIC = 7.8, and MBEC > 1000 µg/mL, using neomycin. Both A. hydrophila and C. portucalensis activated an acyl homoserine lactone (AHL) dependent biosensor, suggesting that quorum sensing could mediate biofilm formation. Their multidrug resistance in the planktonic form, and weak biofilm eradication even with neomycin and cotrimoxazole, indicate that A. hydrophila and C. portucalensis are potential zoonotic pathogens, with risks for patients living with implants.

Detectives and helpers: Natural products as resources for chemical probes and compound libraries.

About 70% of the drugs in use are derived from natural products, either used directly or in chemically modified form. Among all possible small molecules (not greater than 5 kDa), only a few of them are biologically active. Natural product libraries may have a higher rate of finding "hits" than synthetic libraries, even with the use of fewer compounds. This is due to the complementarity between the "chemical space" of small molecules and biological macromolecules such as proteins, DNA and RNA, in addition to the three-dimensional complexity of NPs. Chemical probes are molecules which aid in the elucidation of the biological mechanisms behind the action of drugs or drug-like molecules by binding with macromolecular/cellular interaction partners. Probe development and application have been spurred by advancements in photoaffinity label synthesis, affinity chromatography, activity based protein profiling (ABPP) and instrumental methods such as cellular thermal shift assay (CETSA) and advanced/hyphenated mass spectrometry (MS) techniques, as well as genome sequencing and bioengineering technologies. In this review, we restrict ourselves to a survey of natural products (including peptides/mini-proteins and excluding antibodies), which have been applied largely in the last 5 years for the target identification of drugs/drug-like molecules used in research on infectious diseases, and the description of their mechanisms of action.

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.

Isolation and whole-genome sequencing of Pseudomonas sp. RIT 623, a slow-growing bacterium endowed with antibiotic properties.

OBJECTIVE: There is an urgent need for the discovery and/or development of novel antibiotics. We report an exploration of "slow"-growing bacteria, which can be difficult to isolate using rich media as they are usually outcompeted by "fast"-growing bacteria, as potential sources of novel antimicrobials. RESULTS: Pseudomonas sp. RIT 623 was isolated using pond water agar from a pond located on the campus of the Rochester Institute of Technology (RIT). The genome was sequenced and analyzed for potential secondary metabolite gene clusters. Bioinformatics analysis revealed 14 putative gene clusters predicted to encode pathways for the anabolism of secondary metabolites. Ethyl acetate extracts from spent growth medium of Pseudomonas sp. RIT 623 were tested against two Gram-negative (E. coli ATCC 25922 and P. aeruginosa ATCC 27853) and two Gram-positive (B. subtilis BGSC 168 and S. aureus ATCC 25923) type strains to assess antibiotic activity. The antibiotic assays demonstrated that extracts of Pseudomonas sp. RIT 623 were able to inhibit the growth of the four strains. The active compound was separated using diethyl ether in a multi-solvent extraction and reverse phase chromatography. The bioactive compound/s were subsequently eluted in two consecutive fractions corresponding to approximately 16-22% acetonitrile, indicative of polar compound/s.

Structure-Function Studies of the Antibiotic Target l,l-Diaminopimelate Aminotransferase from Verrucomicrobium spinosum Reveal an Unusual Oligomeric Structure.

While humans lack the biosynthetic pathways for meso-diaminopimelate and l-lysine, they are essential for bacterial survival and are therefore attractive targets for antibiotics. It was recently discovered that members of the Chlamydia family utilize a rare aminotransferase route of the l-lysine biosynthetic pathway, thus offering a new enzymatic drug target. Here we characterize diaminopimelate aminotransferase from Verrucomicrobium spinosum (VsDapL), a nonpathogenic model bacterium for Chlamydia trachomatis. Complementation experiments verify that the V. spinosum dapL gene encodes a bona fide diaminopimelate aminotransferase, because the gene rescues an Escherichia coli strain that is auxotrophic for meso-diaminopimelate. Kinetic studies show that VsDapL follows a Michaelis-Menten mechanism, with a KMapp of 4.0 mM toward its substrate l,l-diaminopimelate. The kcat (0.46 s-1) and the kcat/KM (115 s-1 M-1) are somewhat lower than values for other diaminopimelate aminotransferases. Moreover, whereas other studied DapL orthologs are dimeric, sedimentation velocity experiments demonstrate that VsDapL exists in a monomer-dimer self-association, with a KD2-1 of 7.4 µM. The 2.25 Å resolution crystal structure presents the canonical dimer of chalice-shaped monomers, and small-angle X-ray scattering experiments confirm the dimer in solution. Sequence and structural alignments reveal that active site residues important for activity are conserved in VsDapL, despite the lower activity compared to those of other DapL homologues. Although the dimer interface buries 18% of the total surface area, several loops that contribute to the interface and active site, notably the L1, L2, and L5 loops, are highly mobile, perhaps explaining the unstable dimer and lower catalytic activity. Our kinetic, biophysical, and structural characterization can be used to inform the development of antibiotics.

Creation of an electrokinetic characterization library for the detection and identification of biological cells.

The rising concern over drug-resistant microorganisms has increased the need for rapid and portable detection systems. However, the traditional methods for the analysis of microorganisms can be both resource and time intensive. This contribution presents an alternative approach for the characterization of microorganisms using a microscale electrokinetic technique. The present study aims to develop and validate a library with a novel parameter referred to as the electrokinetic equilibrium condition for each strain, which will allow for fast identification of the studied bacterial and yeast cells in electrokinetic (EK) microfluidic devices. To create the library, experiments with six organisms of interest were conducted using insulator-based EK devices with circle-shaped posts. The organisms included one yeast strain, Saccharomyces cerevisiae; one salmonella strain, Salmonella enterica; two species from the same genus, Bacillus cereus and Bacillus subtilis; and two Escherichia coli strains. The results from these experiments were then analyzed with a mathematical model in COMSOL Multiphysics®, which yielded the electrokinetic equilibrium condition for each distinct strain. Lastly, to validate the applicability EK library, the COMSOL model was used to estimate the trapping conditions needed in a device with oval-shaped posts for each organism, and these values were then compared with experimentally obtained values. The results suggest the library can be used to estimate trapping voltages with a maximum relative error of 12%. While the proposed electrokinetic technique is still a novel approach and the analysis of additional microorganisms would be needed to expand the library, this contribution further supports the potential of microscale electrokinetics as a technique for the rapid and robust characterization of microbes. Graphical abstract.

Whole-Genome Sequencing of Pantoea sp. Strain RIT388, a Potential Oral Opportunistic Pathogen Isolated from a Chewing Stick (Distemonanthus benthamianus).

In this study, we report the isolation, identification, characterization, and whole-genome sequence of the endophyte Pantoea sp. strain RIT388, isolated from Distemonanthus benthamianus, a plant known for its antifungal and antibacterial properties that is commonly used for chewing sticks.

Defeating the trypanosomatid trio: proteomics of the protozoan parasites causing neglected tropical diseases.

Mass spectrometry-based proteomics enables accurate measurement of the modulations of proteins on a large scale upon perturbation and facilitates the understanding of the functional roles of proteins in biological systems. It is a particularly relevant methodology for studying Leishmania spp., Trypanosoma cruzi and Trypanosoma brucei, as the gene expression in these parasites is primarily regulated by posttranscriptional mechanisms. Large-scale proteomics studies have revealed a plethora of information regarding modulated proteins and their molecular interactions during various life processes of the protozoans, including stress adaptation, life cycle changes and interactions with the host. Important molecular processes within the parasite that regulate the activity and subcellular localisation of its proteins, including several co- and post-translational modifications, are also accurately captured by modern proteomics mass spectrometry techniques. Finally, in combination with synthetic chemistry, proteomic techniques facilitate unbiased profiling of targets and off-targets of pharmacologically active compounds in the parasites. This provides important data sets for their mechanism of action studies, thereby aiding drug development programmes.

The Arabidopsis thaliana gene annotated by the locus tag At3g08860 encodes alanine aminotransferase.

The aminotransferase gene family in the model plant Arabidopsis thaliana consists of 44 genes, eight of which are suggested to be alanine aminotransferases. One of the putative alanine aminotransferases genes, At3g08860, was attributed the function of alanine:glyoxylate aminotransferase/ß-alanine:pyruvate aminotransferase based on the analysis of gene expression networks and homology to other ß-alanine aminotransferases in plants. It was earlier demonstrated that At3g08860 is specifically upregulated in response to osmotic stress, but not other stresses (ß-alanine is an osmoprotectant in plants). Furthermore, it was shown that the expression of At3g08860 is highly coordinated with the genes of the uracil degradation pathway leading to the non-proteinogenic amino acid ß-alanine. These evidence were suggestive of the involvement of At3g08860 in ß-alanine metabolism. However, direct experimental evidence for the function of At3g08860 was lacking, and therefore, the goal of this study was to elucidate the function of the uncharacterized aminotransferase annotated by the locus tag At3g08860. The cDNA of At3g08860 was demonstrated to functionally complement two E. coli mutants auxotrophic for the amino acids, L-alanine (proteinogenic) and ß-alanine (non-proteinogenic). Enzyme activity using purified recombinant At3g08860 further demonstrated that the enzyme is endowed with L-alanine:glyoxylate aminotransferase activity.

The Synthesis and Role of ß-Alanine in Plants.

Most studies on amino acids are focused on the proteinogenic amino acids given their essential roles in protein synthesis among other pathways. In addition to 20 ubiquitous amino acids used in protein synthesis, plants synthesize over 250 non-proteinogenic amino acids that are involved in the synthesis of compounds that are anti-herbivory, anti-microbial, response to abiotic stresses, nitrogen storage, toxins against both vertebrates/invertebrates, and plant hormones among others. One such non-proteinogenic acid is ß-alanine, which is known mainly for studies on humans. ß-Alanine forms a part of pantothenate (vitamin B5), which is incorporated into the universal carbon shuttling compounds Coenzyme A and acyl carrier protein, in all organisms including plants. The focus of this review, however, is on the biosynthesis, metabolism, and the role of ß-alanine in plants. There are several functions of ß-alanine unique to plants. It is accumulated as a generic stress response molecule involved in protecting plants from temperature extremes, hypoxia, drought, heavy metal shock, and some biotic stresses. There is evidence of its participation in lignin biosynthesis and ethylene production in some species. It is further converted to the osmoprotective compound ß-alanine betaine in some species and converted to the antioxidant homoglutathione in others. The polyamines spermine/spermidine, propionate and uracil have been shown to be precursors of ß-alanine in plants. However, plants vary in terms of their biosynthetic pathways, and the primary metabolism of ß-alanine is far from settled.