Applied and Environmental Microbiology最新文献

In motor-assisted nanopore sensing, increasing the salt concentration improves the signal-to-noise ratio (SNR); however, helicases used as motor proteins generally fail to sustain efficient unwinding under high-salt conditions. In this study, we characterized the structure and function of AAA+ ATPase helicases from acidophiles. Bioinformatic analysis indicated that such helicases are broadly distributed among acidophilic bacteria. A representative helicase from Leptospirillum spp., designated LfDda, adopts a dimeric architecture at 3.5 Å resolution with a flexible tower domain while retaining conserved 1A and 2A domains, akin to well-studied motor (T4 Dda helicase). Biochemical assays demonstrated that the dimeric form of LfDda is functional, acting as a 5'-3' directional helicase that can utilize various divalent metal ions as cofactors, with Mn²⁺ supporting the highest catalytic activity. Crosslinking of the flexible 1B and 2B domains yielded a monomeric variant capable of efficient DNA translocation at 600 mM KCl, representing a 6-fold enhancement in ionic tolerance. These findings suggest that helicases derived from extremophilic bacteria can be engineered to modulate DNA translocation behavior, providing a potential avenue for developing new motor proteins for nanopore and other DNA-unwinding applications.

Importance: Nanopore sensing is a powerful approach for detecting and analyzing DNA at the single-molecule level, but its performance relies on specialized motor proteins that control DNA movement. A major challenge is that most available helicases lose activity in the high-salt conditions required to enhance signal quality. In this study, we characterized and engineered a helicase from an acidophilic bacterium that naturally thrives in extreme environments. By resolving its structure and stabilizing its flexible domains, we created a variant that remains functional under salt levels where conventional helicases fail, achieving a 6-fold increase in tolerance. These findings highlight extremophile enzymes as promising resources for designing robust molecular motors, expanding the toolbox for nanopore-based sensing and related biotechnological applications.

In recent decades, many researchers have aimed to create suppressive composts and determine the factors that distinguish them from non-suppressive ones. The research conducted by A. Logo, B. Boppré, J. Fuchs, M. Maurhofer, et al. (Appl Environ Microbiol 91:e01100-25, 2025, https://doi.org/10.1128/aem.01100-25) represents a significant advancement in understanding the microbiological bases of this phenomenon, as it evaluates the suppressiveness of 37 composts across three different pathosystems. The authors successfully identified certain bacterial and fungal taxa that could potentially act as indicators of specific disease suppressiveness.

Piscirickettsia salmonis is the causative agent of salmonid rickettsial septicemia (SRS), the main bacterial disease affecting the salmon industry in Chile. In this work, we implemented a Mobile-CRISPRi system to generate gene silencing using a catalytically inactive dCas9 protein and an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible single-guide RNA (sgRNA). We demonstrate the efficacy of the CRISPRi system in P. salmonis by silencing an exogenous reporter (sfGFP) and an endogenous regulator (Fur) that controls intracellular iron homeostasis in bacteria. The inducible expression of dCas9 and the sfGFP-directed sgRNA caused a 98.7% decrease in fluorescence in the knockdown strain. This silencing system was effective in seven P. salmonis strains from both genogroups. Furthermore, the same system was used to construct fur knockdown strains. A 50-fold decrease in fur expression level was determined in these strains when the expression of the fur gRNA was induced with IPTG. By RNA-seq, we detected a significant increase in the expression of genes encoding the Fe²⁺ and Fe³⁺ acquisition systems and iron mobilization in the fur1 knockdown after IPTG induction. All the genes with over 2-fold increased expression in the RNA-seq presented the Fur box consensus sequence in their regulatory region. The implementation of the Mobile-CRISPRi system in P. salmonis has been demonstrated to be effective, thus providing a tool with potential application for the analysis of gene function in this pathogen. It is anticipated that these analyses will be valuable in identifying genes involved in the mechanisms of pathogenesis of P. salmonis.

Importance: Salmonid rickettsial septicemia (SRS) is an infectious disease caused by the marine bacterium Piscirickettsia salmonis. This Gamma-proteobacteria is a fastidious and facultative intracellular pathogen that has a nearly worldwide distribution, particularly impacting Chilean salmonid aquaculture. Its fastidious nature has made it hard to grow in labs, hindering research into its virulence and treatment, especially because of the lack of molecular techniques to study gene function. We show here the successful implementation of the Mobile-CRISPRi system for gene silencing. Significantly, we have adapted this technique for use with the marine pathogen P. salmonis, inserting exogenous genes into the bacterium's chromosome to ensure their constitutive and inducible expression and silencing both exogenous and endogenous gene expression. The Mobile-CRISPRi system was also used to study the iron regulator Fur, confirming Fur's relevance to the iron metabolism in the pathogen.

Amino acid racemases are pivotal for d-amino acid (DAA) biosynthesis with wide-ranging biotechnological applications, yet their industrial deployment is hindered by narrow substrate specificity and instability. Here, we report the discovery of Halocola ammonii gen. nov., sp. nov. DA487^T, a novel taxon within the proposed family Halocolacceae fam. nov. (order Flavobacteriales), isolated from hypersaline sediments. Genomic analysis revealed a robust DAA metabolic network, including a putative broad-specificity racemase RacX. Biochemical characterization demonstrated RacX's exceptional catalytic efficiency (k_cat/K_m = 151.2 s^-1 mM^-1 for l-Lys, k_cat/K_m = 17.8 s^-1 mM^-1 for d-Lys) and broad substrate spectrum (15/17 tested l-amino acids). Homology modeling and mutagenesis identified Ala79 and Cys193 as putative catalytic residues, based on structural conservation with EcL-DER. Remarkably, the A79C variant enhanced the reverse reaction efficiency (d-Lys → l-Lys) by 44%, effectively shifting the enzyme's catalytic bias and the resulting steady-state ratio of enzyme-bound species. Computational docking suggested that Asn80, Thr81, Asn121, and Thr124 may modulate substrate binding, though experimental structural validation is required. The thermostability-lability tradeoff ([Formula: see text]) highlights targets for protein engineering. Our findings not only expand the phylogenetic diversity of microbial racemases but also identify a promising biocatalyst candidate for industrial DAA production.IMPORTANCEMicrobial adaptations to extreme environments serve as a valuable source of novel biocatalysts with potential for sustainable industrial applications. In this study, we characterized Halocola ammonii DA487ᵀ, a halophilic bacterium representing the novel family Halocolaceae within the order Flavobacteriales, and identified a broad-specificity amino acid racemase, RacX. RacX demonstrates exceptional catalytic efficiency (k_cat/K_m up to 151.2 s⁻¹ mM⁻¹ for l-Lys) across multiple amino acids and exhibits remarkable stability under neutral and alkaline conditions (pH 7.0-9.0)-properties intrinsically linked to its high-salt ecological niche. Unlike most known racemases from neutrophilic organisms, RacX originates from an understudied phylogenetic lineage and displays unique mechanistic features, including a strong innate bias toward d-amino acid (DAA) production that can be rationally reprogrammed via single-residue substitution (e.g., A79C). These functional and evolutionary insights, combined with its halotolerance and broad substrate scope, position RacX as a promising and engineerable biocatalyst for industrial processes requiring operation under high-salt or alkaline conditions, such as the synthesis of DAA precursors for antibiotics.

Marine environments are frequently oligotrophic, characterized by low amount of bioassimilable nitrogen sources. At the global scale, the microbial fixation of N₂, or diazotrophy, represents the primary source of fixed nitrogen in pelagic marine ecosystems, playing a key role in supporting primary production and driving the export of organic matter to the deep ocean. However, given the high energetic cost of N₂ fixation, the active release of fixed nitrogen by diazotrophs appears counterintuitive, suggesting the existence of alternative passive release pathways that remain understudied to date. Here, we show that the marine non-cyanobacterial diazotroph Vibrio diazotrophicus is endowed with a prophage belonging to the Myoviridae family, whose expression is induced under anoxic and biofilm-forming conditions. We demonstrate that this prophage can spontaneously excise from the genome of its host and that it forms intact and infective phage particles. Moreover, phage-mediated host cell lysis leads to increased biofilm production compared with a prophage-free derivative mutant and to increased release of dissolved organic carbon and ammonium. Altogether, the results suggest that viruses may play a previously unrecognized role in oceanic ecosystem dynamics by structuring microhabitats suitable for diazotrophy and by contributing to the recycling of (in)organic matter.

Importance: Diazotrophs are key players in ocean functioning by providing fixed nitrogen to ecosystems and fueling primary production. However, from a physiological point of view, the active release of nitrogenous compounds by diazotrophs is paradoxical, since they would invest in an energy-intensive process and supply nutrient to non-sibling cells, with the risk of being outcompeted. Therefore, alternative ways leading to the release of fixed nitrogen must exist. Here, we show that the marine non-cyanobacterial diazotroph Vibrio diazotrophicus possesses one prophage, whose activation leads to cell death, increased biofilm production, and the release of dissolved organic compounds and ammonium. Taken together, our results provide evidence that marine phage-diazotroph interplay leads to the creation of microhabitats suitable for diazotrophy, such as biofilm, and to nutrient cycling, and contributes to better understanding of the role of viruses in marine ecosystems.

Biofouling presents significant challenges to the crop production industry, notably reducing irrigation efficiency and potentially dispersing pathogens to irrigated crops. This study evaluated the efficacy of peracetic acid (PAA) and chlorine (Cl) against Salmonella biofilms in irrigation lines with or without fertilizers. Pond water (PW) with 2-4-1 fish emulsion (O), PW with 4-0-8 synthetic liquid fertilizer (S), or PW with no fertilizer (NoFert) was inoculated with 2 log CFU/mL of a rifampicin-resistant Salmonella cocktail. Inoculated water was then circulated through polyethylene loop irrigation system for a month. Salmonella populations both in the water and attached to the tubing were determined. Data showed that a single point of contamination from the water resulted in a biofilm formation with O and NoFert, but not the S treatments, after 3 days. Both PAA and Cl effectively reduced Salmonella populations for all fertilizer treatments in water samples. However, when no sanitizer was introduced to the line, bacterial dispersion resulted in the contamination of a subsequent irrigation event for the O treatments but not the S and NoFert treatments, which presented no microbial proliferation. Our findings suggest that O treatments resulted in persistent biofilm formation that could lead to contamination of irrigation water when no sanitizers are introduced. These studies provide insight into the behavior of foodborne pathogens in irrigation distribution systems.IMPORTANCEThe accumulation of bacteria in water distribution systems due to biofouling can lead to contamination, making it crucial to evaluate and implement effective mitigation measures to prevent these issues and ensure safe and efficient irrigation practices. The use of the 2-4-1 fish emulsion in-line may support the establishment of Salmonella biofilms and subsequent cross-contamination of irrigation water if not fully flushed from the system. This study demonstrates that PAA and Cl effectively reduce Salmonella contamination in water but will not eliminate populations in-line once biofilms are established.