Publications

4495

Abstract Aims Huanglongbing (citrus greening) is a plant disease putatively caused by the unculturable Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), and it has caused severe damage to citrus plantations worldwide. There are no definitive treatments for this disease, and conventional disease control techniques have shown limited efficacy. This work presents an in silico evaluation of using specifically targeting anti-microbial peptides (STAMPs) consisting of a targeting segment and an antimicrobial segment to inhibit citrus greening by inhibiting the BamA protein of CLas, which is an outer membrane protein crucial for bacterial viability. Methods and results Initially, a set of peptides with a high affinity toward BamA protein were screened and evaluated via molecular docking and molecular dynamics simulations and were verified in vitro via bio-layer interferometry (BLI). In silico studies and BLI experiments indicated that two peptides, HASP2 and HASP3, showed stable binding to BamA. Protein structures for STAMPs were created by fusing known anti-microbial peptides (AMPs) with the selected short peptides. The binding of STAMPs to BamA was assessed using molecular docking and binding energy calculations. The attachment of high-affinity short peptides significantly reduced the free energy of binding for AMPs, suggesting that it would make it easier for the STAMPs to bind to BamA. Efficacy testing in vitro using a closely related CLas surrogate bacterium showed that STAMPs had greater inhibitory activity than AMP alone. Conclusions In silico and in vitro results indicate that the STAMPs can inhibit CLas surrogate Rhizobium grahamii more effectively compared to AMPs, suggesting that STAMPs can achieve better inhibition of CLas, potentially via enhancing the site specificity of AMPs.

Abstract The Candidate Phyla Radiation (CPR) encompasses widespread uncultivated bacteria with reduced genomes and limited metabolic capacities. Most CPR bacteria lack the minimal set of enzymes required for peptidoglycan (PG) synthesis, leaving it unclear how these bacteria produce this essential envelope component. In this study, we analysed the distribution of d ‐amino acid racemases that produce the universal PG components d ‐glutamate ( d ‐Glu) or d ‐alanine ( d ‐Ala). We also examined moonlighting enzymes that synthesize d ‐Glu or d ‐Ala. Unlike other phyla in the domain Bacteria, CPR bacteria do not exhibit these moonlighting activities and have, at most, one gene encoding either a Glu or Ala racemase. One of these ‘orphan’ racemases is a predicted Glu racemase (MurI CPR ) from the CPR bacterium Candidatus Saccharimonas aalborgenesis . The expression of MurI CPR restores the growth of a Salmonella d ‐Glu auxotroph lacking its endogenous racemase and results in the substitution of l ‐Ala by serine as the first residue in a fraction of the PG stem peptides. In vitro, MurI CPR exclusively racemizes Glu as a substrate. Therefore, Ca. Saccharimonas aalborgensis may couple Glu racemization to serine and d ‐Glu incorporation into the stem peptide. Our findings provide the first insights into the synthesis of PG by an uncultivated environmental bacterium and illustrate how to experimentally test enzymatic activities from CPR bacteria related to PG metabolism.

Huanglongbing (HLB) is a disease caused by the phloem- limited Candidatus Liberibacter asiaticus (CLas) that affects the citrus industry worldwide. To date, only indirect strategies have been implemented to eradicate HLB. Included among these is the population control of the psyllid vector (Diaphorina citri), which usually provides inconsistent results. Even though strategies for direct CLas suppression seem a priori more promising, only a handful of reports have been focused on a confrontation of the pathogen. Recent developments in polymer chemistry have allowed the design of polycationic self-assembled block copolymers with outstanding antibacterial capabilities. Here, we report the use of polymeric nano-sized bactericide particles (PNB) to control CLas directly in the phloem vasculature. The field experiments were performed in Rioverde, San Luis Potosí, and is one of the most important citrusproducing regions in Mexico. An average 52% reduction in the bacterial population was produced when PNB was injected directly into the trunk of 20 infected trees, although, in some cases, reduction levels reached 97%. These results position PNB as a novel and promising nanotechnological tool for citrus crop protection against CLas and other related pathogens.

AbstractFive cyanobacterial strains exhibiting Nostoc‐like morphology were sampled from the biodiversity hotspots of the northeast region of India and characterized using a polyphasic approach. Molecular and phylogenetic analysis using the 16S rRNA gene indicated that the strains belonged to the genera Amazonocrinis and Dendronalium. In the present investigation, the 16S rRNA gene phylogeny clearly demarcated two separate clades of Amazonocrinis. The strain MEG8‐PS clustered along with Amazonocrinis nigriterrae CENA67, which is the type strain of the genus. The other three strains ASM11‐PS, RAN‐4C‐PS, and NP‐KLS‐5A‐PS clustered in a different clade that was phylogenetically distinct from the Amazonocrinis sensu stricto clade. Interestingly, while the 16S rRNA gene phylogeny exhibited two separate clusters, the 16S–23S ITS region analysis did not provide strong support for the phylogenetic observation. Subsequent analyses raised questions regarding the resolving power of the 16S–23S ITS region at the genera level and the associated complexities in cyanobacterial taxonomy. Through this study, we describe a novel genus Ahomia to accommodate the members clustering outside the Amazonocrinis sensu stricto clade. In addition, we describe five novel species, Ahomia kamrupensis, Ahomia purpurea, Ahomia soli, Amazonocrinis meghalayensis, and Dendronalium spirale, in accordance with the International Code of Nomenclature for algae, fungi, and plants (ICN). Apart from further enriching the genera Amazonocrinis and Dendronalium, the current study helps to resolve the taxonomic complexities revolving around the genus Amazonocrinis and aims to attract researchers to the continued exploration of the tropical and subtropical cyanobacteria for interesting taxa and lineages.

Ciliates are unicellular eukaryotes, regularly involved in symbiotic associations. Symbionts may colonize the inside of their cells as well as their surface as ectosymbionts. Here, we report on a new ciliate species, designated asZoothamnium mariellasp. nov. (Peritrichia, Sessilida), discovered in the northern Adriatic Sea (Mediterranean Sea) in 2021. We found this ciliate species to be monospecifically associated with a new genus of ectosymbiotic bacteria, here proposed asCandidatusFusimicrobium zoothamnicola gen. nov., sp. nov. To formally describe the new ciliate species, we investigated its morphology and sequenced its 18S rRNA gene. To demonstrate its association with a single species of bacterial ectosymbiont, we performed 16S rRNA gene sequencing, fluorescencein situhybridization, and scanning electron microscopy. Additionally, we explored the two partners’ cultivation requirements and ecology.Z. mariellasp. nov. was characterized by a colony length of up to 1 mm. A consistent number of either seven or eight long branches alternated on the stalk in close distance to each other. The colony developed three different types of zooids: microzooids (“trophic stage”), macrozooids (“telotroch stage”), and terminal zooids (“dividing stage”). Viewed from inside the cell, the microzooids’ oral ciliature ran in 1 ¼ turns in a clockwise direction around the peristomial disc before entering the infundibulum, where it performed another ¾ turn. Phylogenetic analyses assignedZ. mariellasp. nov. to clade II of the family Zoothamnidae. The ectosymbiont formed a monophyletic clade within theGammaproteobacteriaalong with two other ectosymbionts of peritrichous ciliates and a free-living vent bacterium. It colonized the entire surface of its ciliate host, except for the most basal stalk of large colonies, and exhibited a single, spindle-shaped morphotype. Furthermore, the two partners together appear to be generalists of temperate, oxic, marine shallow-water environments and were collectively cultivable in steady flow-through systems.