Applied and Environmental Microbiology最新文献

Flavobacterium psychrophilum causes bacterial cold-water disease (BCWD) in salmonids and other fish, resulting in substantial economic losses in aquaculture worldwide. The mechanisms F. psychrophilum uses to cause disease are poorly understood. Despite considerable effort, most strains of F. psychrophilum have resisted attempts at genetic manipulation. F. psychrophilum restriction-modification (R-M) systems may contribute to this resistance. Restriction endonucleases (REases) rapidly degrade nonself DNA if it is not properly methylated by their cognate DNA methyltransferases (MTases). We used comparative genomics to show that R-M systems are abundant in F. psychrophilum and that strain-specific variations partially align with phylogeny. We identified two critical type II R-M systems, HpaII-like (FpsJI) and ScrFI-like (FpsJVI), that are conserved in most of the sequenced strains. Protection of foreign DNA against HpaII and ScrFI was achieved by expression of the MTases M.FpsJI and M.FpsJVI in Escherichia coli. Furthermore, deleting the two REase genes from F. psychrophilum resulted in efficient conjugative DNA transfer from E. coli into the otherwise genetically intractable F. psychrophilum strain CSF259-93. This allowed us to construct a CSF259-93 mutant lacking gldN, a core component of the type IX protein secretion system. The pre-methylation system developed in this study functions in all tested F. psychrophilum strains harboring HpaII-like and ScrFI-like REases. These newly developed genetic tools may allow the identification of key virulence factors and facilitate the development of live attenuated vaccines or other measures to control BCWD.

Importance: Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum is a problem for salmonid aquaculture worldwide, and current control measures are inadequate. An obstacle in understanding and controlling BCWD is that most F. psychrophilum strains resist DNA transfer, thus limiting genetic studies of their virulence mechanisms. F. psychrophilum restriction enzymes that destroy foreign DNA were suspected to contribute to this problem. Here, we used F. psychrophilum DNA methyltransferases to modify and protect foreign DNA from digestion. This allowed efficient conjugative DNA transfer into nine diverse F. psychrophilum strains that had previously resisted DNA transfer. Using this approach, we constructed a gene deletion mutant that failed to cause disease in rainbow trout. Further genetic studies could help determine the molecular factors involved in pathogenesis and may aid development of innovative BCWD control strategies.

Antimicrobial resistance is an ever-increasing problem for human health, and with only a few novel antimicrobials discovered in recent decades, an extraordinary effort is needed to circumvent this crisis. A promising source of new microbial-derived antimicrobial compounds resides in the large fraction of microbes that are not readily cultured by standard cultivation. It has previously been shown that nests of the social spider Stegodyphus dumicola contain a diverse bacterial community, where only a small fraction of the microbes could be recovered by standard cultivation. To improve the recovery of the bacterial diversity cultured from nests, we modified the previously described isolation chip (iChip) to fit the natural arid environment of S. dumicola nests. Here we provide a comprehensive analysis of the modified iChip's performance. We found that the modified iChip improved the overall culturability, performed equally or better at recovering the bacterial diversity from individual nests, and improved the recovery of rare isolates compared to standard cultivation. Furthermore, we show that the modified iChip can be used in the field. In addition, we observed that the nests contain volatile organic compounds (VOCs) that could serve as substrate for the selective enrichment of rare and iChip-specific isolates. Our modified iChip can be applied for in situ cultivation in a broad range of arid habitats that can be exploited for future drug discovery.IMPORTANCEThe demand for novel antimicrobial compounds is an ever-increasing problem due to the rapid spread of antibiotic-resistant microbes. Therefore, exploring new habitats for microbial-derived antimicrobial compounds is crucial. The nest microbiome of Stegodyphus dumicola remains largely unexplored and could potentially serve as a new source of antimicrobial compounds. To access the nest's microbial diversity, we designed a modified iChip for in situ cultivation inside spider nests and tested its applications in both field and laboratory settings. Our study shows that the iChip's ability to recover in situ abundant genera was comparable or superior to standard cultivation, while the recovery of rare (low-abundant genera) was higher. We argue that these low-abundant and iChip-specific isolates are enriched from naturally occurring nest volatile organic compounds (VOCs) during iChip incubation.

The Winam Gulf in the Kenyan region of Lake Victoria experiences prolific, year-round cyanobacterial harmful algal blooms (cyanoHABs) which pose threats to human, livestock, and ecosystem health. To our knowledge, there is limited molecular research on the gulf's cyanoHABs, and thus, the strategies employed for survival and proliferation by toxigenic cyanobacteria in this region remain largely unexplored. Here, we used metagenomics to analyze the Winam Gulf's cyanobacterial composition, function, and biosynthetic potential. Dolichospermum was the dominant bloom-forming cyanobacterium, co-occurring with Microcystis at most sites. Microcystis and Planktothrix were more abundant in shallow and turbid sites. Metagenome-assembled genomes (MAGs) of Dolichospermum harbored nitrogen fixation genes, suggesting diazotrophy as a potential mechanism supporting the proliferation of Dolichospermum in the nitrogen-limited gulf. Over 300 biosynthetic gene clusters (BGCs) putatively encoding the synthesis of toxins and other secondary metabolites were identified across the gulf, even at sites where there were no visible cyanoHAB events. Almost all BGCs identified had no known synthesis product, indicating a diverse and novel biosynthetic repertoire capable of synthesizing harmful or potentially therapeutic metabolites. Microcystis MAGs contained mcy genes encoding the synthesis of hepatotoxic microcystins which are a concern for drinking water safety. These findings illustrate the spatial variation of bloom-forming cyanobacteria in the Winam Gulf and their available strategies to dominate different ecological niches. This study underscores the need for further use of genomic techniques to elucidate the dynamics and mitigate the potentially harmful effects of cyanoHABs and their associated toxins on human, environmental, and economic health.

Ocean warming due to climate change endangers coral reefs, and regional nitrogen overloading exacerbates the vulnerability of reef-building corals as the dual stress disrupts coral-Symbiodiniaceae mutualism. Different forms of nitrogen may create different interactive effects with thermal stress, but the underlying mechanisms remain elusive. To address the gap, we measured and compared the physiological and transcriptional responses of the Symbiodiniaceae Cladocopium goreaui to heat stress (31°C) when supplied with different types of nitrogen (nitrate, ammonium, or urea). Under heat stress (HS), cell proliferation and photosynthesis of C. goreaui declined, while cell size, lipid storage, and total antioxidant capacity increased, both to varied extents depending on the nitrogen type. Nitrate-cultured cells exhibited the most robust acclimation to HS, as evidenced by the fewest differentially expressed genes (DEGs) and less ROS accumulation, possibly due to activated nitrate reduction and enhanced ascorbate biogenesis. Ammonium-grown cultures exhibited higher algal proliferation and ROS scavenging capacity due to enhanced carotenoid and ascorbate quenching, but potentially reduced host recognizability due to the downregulation of N-glycan biosynthesis genes. Urea utilization led to the greatest ROS accumulation as genes involved in photorespiration, plant respiratory burst oxidase (RBOH), and protein refolding were markedly upregulated, but the greatest cutdown in photosynthate potentially available to corals as evidenced by photoinhibition and selfish lipid storage, indicating detrimental effects of urea overloading. The differential warming nitrogen-type interactive effects documented here has significant implication in coral-Symbiodiniaceae mutualism, which requires further research.IMPORTANCERegional nitrogen pollution exacerbates coral vulnerability to globally rising sea-surface temperature, with different nitrogen types exerting different interactive effects. How this occurs is poorly understood and understudied. This study explored the underlying mechanism by comparing physiological and transcriptional responses of a coral symbiont to heat stress under different nitrogen supplies (nitrate, ammonium, and urea). The results showed some common, significant responses to heat stress as well as some unique, N-source dependent responses. These findings underscore that nitrogen eutrophication is not all the same, the form of nitrogen pollution should be considered in coral conservation, and special attention should be given to urea pollution.

Antimicrobial peptides (AMPs) have emerged as potential alternatives to conventional antibiotics due to their novelty and multiple mechanisms of action. Because they are peptides, AMPs are amenable to bioengineering and suitable for cloning and expression at large production scales. However, the efficient delivery of AMPs is an unaddressed issue, particularly due to their large size, possible toxicities, and the development of adverse immune responses. Here, we have reviewed the possibilities of adeno-associated virus (AAV)-based localized gene delivery of AMPs for the treatment of infectious diseases with a special focus on respiratory infections. By discussing the gene delivery mechanism of AAV and the accompanying technical and therapeutic challenges with AMPs, we describe a foundation that emphasizes the use of viral vector-based gene delivery of AMPs for disease treatment.

Thermodynamics has long been applied in predicting undiscovered microorganisms or analyzing energy flows in microbial metabolism, as well as evaluating microbial impacts on global element distributions. However, further development and refinement in this interdisciplinary field are still needed. This work endeavors to develop a whole-cycle framework integrating thermodynamics with microbiological studies, focusing on representative nitrogen-transforming microorganisms. Three crucial concepts (reaction favorability, energy balance, and reaction directionality) are discussed in relation to nitrogen-transforming reactions. Specifically, reaction favorability, which sheds lights on understanding the diversity of nitrogen-transforming microorganisms, has also provided guidance for novel bioprocess development. Energy balance, enabling the quantitative comparison of microbial energy efficiency, unravels the competitiveness of nitrogen-transforming microorganisms under substrate-limiting conditions. Reaction directionality, revealing the niche-differentiating patterns of nitrogen-transforming microorganisms, provides a foundation for predicting biogeochemical reactions under various environmental conditions. This review highlights the need for a more comprehensive integration of thermodynamics in environmental microbiology, aiming to comprehensively understand microbial impacts on the global environment from micro to macro scales.

Pseudoalteromonas are widely distributed in marine extreme habitats and exhibit diverse extracellular protease activity, which is essential for marine biogeochemical cycles. However, our understanding of viruses that infect Pseudoalteromonas remains limited. This study isolated a virus infecting Pseudoalteromonas nigrifaciens from Xiaogang in Qingdao, China. vB_PunP_Y3 comprises a linear, double-strand DNA genome with a length of 48,854 bp, encoding 52 putative open reading frames. Transmission electron microscopy demonstrates the short-tailed morphology of vB_PunP_Y3. Phylogenetic and genome-content-based analysis indicate that vB_PunP_Y3 represents a novel virus family named as Poorviridae, along with three high-quality uncultivated viral genomes. Biogeographical analyses show that Poorviridae is distributed across five viral ecological zones, and is predominantly detected in the Antarctic, Arctic, and bathypelagic zones. Comparative genomics analyses identified three of the seven hallmark proteins of N4-like viruses (DNA polymerase, major capsid protein, and virion-encapsulated RNA polymerase) from vB_PunP_Y3, combing with the protein tertiary structures of the major capsid protein, suggesting that vB_PunP_Y3 might evolve from the N4-like viruses.

Importance: vB_PunP_Y3 is a unique strain containing three of the seven hallmark proteins of N4-like viruses, but is grouped into a novel family-level viral cluster with three uncultured viruses from metagenomics, named Poorviridae. This study enhanced the understanding about the genetic diversity, evolution, and distribution of Pseudoalteromonas viruses and provided insights into the novel evolution mechanism of marine viruses.

Sourdoughs are maintained by back-slopping over long time periods. To determine strain-level persistence of bacteria, we characterized four sourdoughs from bakeries over a period of 3.3, 11.0, 18.0, and 19.0 years. One sourdough included isolates of Levilactobacillus spp. and Fructilactobacillus spp. that differed by fewer than 10 single-nucleotide polymorphisms (SNPs) from the isolates obtained 3.3 years earlier and thus likely represent the same strain. Isolates of Levilactobacillus parabrevis differed by 200-300 SNPs; their genomes were under positive selection, indicating transmission from an external source. In two other sourdoughs, isolates of Fructilactobacillus sanfranciscensis that were obtained 11 and 18 years apart differed by 19 and 29 SNPs, respectively, again indicating repeated isolation of the same strain. The isolate of Fl. sanfranciscensis from the fourth sourdough differed by 45 SNPs from the isolate obtained 19 years previously. We thus identified strain-level persistence in three out of four long-term back-slopped sourdoughs, making it possible that strains persisted over periods that are long enough to allow bacterial speciation and domestication.IMPORTANCEThe assembly of microbial communities in sourdough is shaped by dispersal and selection. Speciation and domestication of fermentation microbes in back-slopped food fermentations have been documented for food-fermenting fungi including sourdough yeasts but not for bacteria, which evolve at a slower rate. Bacterial speciation in food fermentations requires strain-level persistence of fermentation microbes over hundreds or thousands of years. By documenting strain-level persistence in three out of four sourdoughs over a period of up to 18 years, we demonstrate that persistence over hundreds or thousands of years is possible, if not likely. We thus not only open a new perspective on fermentation control in bakeries but also support the possibility that all humans, despite their cultural diversity, share the same fermentation microbes.

Wastewater surveillance for infectious agents has proved useful in identifying the circulation of viruses within populations. We investigated the presence and concentration of human immunodeficiency virus (HIV)-1 total nucleic acids (including both viral RNA and proviral DNA) in wastewater solids. We retrospectively measured HIV-1 nucleic acids in two samples per week for 26 months at two wastewater treatment plants serving populations with different prevalences of HIV infections in San Francisco and Santa Clara County, California, USA. We detected HIV nucleic acids in a majority of samples with concentrations ranging from non-detect to 3.9 × 10⁵ cp/g (N = 459 samples total). Concentrations of HIV-1 were significantly higher in samples from the wastewater treatment plant serving a population with a higher prevalence of people living with HIV than in the plant serving a population with a lower prevalence. The HIV-1 nucleic acids amplified were primarily DNA and thus represented proviral DNA shedding into wastewater. Additionally, we found that HIV-1 nucleic acid concentrations in wastewater solids were orders of magnitude higher than those in liquid wastewater indicating that the HIV-1 target preferentially sorbs to solids. Whether concentrations of HIV-1 in wastewater solids can be used to identify the number of incident cases remains unknown. Additional work on HIV-1 shedding from individuals with viremia and people living with HIV is needed to translate wastewater measurements into quantitative information on infections. Additional work may also be needed to document non-human sources of HIV-1 nucleic acids in wastewater.

Importance: Human immunodeficiency virus (HIV)-1 has infected nearly 100 million people since it emerged in the 1980s. Antiretroviral therapy prevents transmission of HIV and also allows infected individuals to live healthy lives with normal life expectancy. Consequently, identifying unrecognized cases of HIV is of paramount importance. Since wastewater represents a composite biological sample from a community, it may provide valuable information on HIV-1 prevalence that can be used to inform HIV testing and outreach.