Applied and Environmental Microbiology最新文献

Because of the urgent need for new antibiotics to treat drug-resistant bacterial pathogens, we employed an assay that rapidly screens large quantities of compounds for their ability to interfere with bacterial protein synthesis, in particular, the delivery of amino acids to the ribosome via tRNA and elongation factor Tu (EF-Tu). We have identified a drug lead, named MGC-10, which kills Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), with a MIC of 6 µM, while being harmless to mammalian cells in vitro in that concentration range. The antibacterial activity of MGC-10 was broad against over 50 strains of antibiotic-resistant samples obtained from hospital infections, where MGC-10 inhibited all tested strains of MRSA. Extensive selection and screening with MGC-10 did not yield any resistant strains, indicating it may have universal antibacterial activity against S. aureus. Pharmacokinetics performed in mice suggested that MGC-10 was too toxic for systemic use; however, it appears to have potential as a topical treatment for difficult-to-treat wounds or skin infections by Gram-positive pathogens such as MRSA. In a mouse skin-infection model with MRSA, MGC-10 performed as well or better than the present topical drug of choice, mupirocin. MGC-10 showed little, if any, accumulation in the livers of topically treated mice. These results bode well for the future use of MGC-10 in clinical application as it could be used to treat a broad range of S. aureus skin infections that are resistant to known antibiotics.IMPORTANCEThere is a critical need for new antibiotics to treat bacterial infections caused by pathogens resistant to many if not all currently available antibiotics. We describe here the identification of a prospective new antibiotic from high-throughput screening of a chemical library. The screening was designed to detect the inhibition of formation of a complex required for bacterial protein synthesis in all bacteria, the "ternary complex," comprised of elongation factor Tu (EF-Tu), aminoacyl-tRNA, and GTP. The inhibitory compound, renamed MGC-10, was effective against all Gram-positive bacteria, including a wide variety of methicillin-resistant Staphylococcus aureus (MRSA) strains. Although apparently too toxic for systemic use, the compound was safe and effective for topical use for treating skin infections in a mouse model. No resistance to the compound has been detected thus far, suggesting the potential to develop this compound for topical use to treat infections, especially those caused by pathogens resistant to existing antibiotics.

The overuse and wanton discharge of antibiotics produces a threat to bacteria in the environment, which, in turn, stimulates the more rapid emergence of antibiotic-resistant bacteria. Pseudomonas putida actively forms biofilms to protect the population under tetracycline stress, but the molecular mechanism remains unclear. This study found that tetracycline at sub-minimal inhibitory concentrations increased cyclic diguanylate (c-di-GMP), a second messenger that positively regulates biofilm formation. Four c-di-GMP-metabolizing proteins were found to be involved in the tetracycline-mediated biofilm promotion, including DibA, WspR, PP_3242, and PP_3319. Among them, the diguanylate cyclase WspR displayed the most significant effect on c-di-GMP level and biofilm formation. WspR belongs to the wsp operon comprising seven genes (wspA-wspF and wspR). The wsp operon contained six promoters, including one major start promoter (P_wspA) and five internal promoters (P_wspB, P_wspC, P_wspD, P_wspF, and P_wspR), and tetracycline promoted the activity of P_wspA. The stress-response sigma factor RpoS directly bound to P_wspA and positively regulated its activity under tetracycline stress. Moreover, RpoS was required for tetracycline to induce P_wspA activity and promote biofilm formation. Our results enrich the transcriptional regulation of the wsp operon and reveal the mechanism by which tetracycline promotes biofilm formation in P. putida.IMPORTANCEThe overuse and wanton discharge of antibiotics produces a threat to bacteria in the environment, which, in turn, stimulates the more rapid emergence of antibiotic-resistant bacteria. The Pseudomonas putida actively forms biofilm against antibiotic threats, but the mechanism remains unclear. Here, our results showed that tetracycline treatment at sub-minimal inhibitory concentrations could induce the expression of the Wsp system via the sigma factor RpoS in P. putida, resulting in elevated c-di-GMP levels, which leads to increased biofilm formation. The wsp operon contains one major promoter and five internal promoters, and RpoS directly binds to the major promoter to promote its activity.

Arbuscular mycorrhizal fungi (AMF) are promoted as commercial bioinoculants for sustainable agriculture. Little is known, however, about the survival of AMF inoculants in soil and their impacts on native or pre-established AMF communities in root tissue. The current study was designed to assess the stability of pre-existing/nursery-derived AMF in apple rootstocks after being planted into soil containing a known community of AMF with a limited number of species. Root-associated endophytic communities (bacteria and fungi) are known to differ depending on apple rootstock genotype. Thus, an additional aim of this study was to explore the effect of apple rootstock genotype on AMF community structure. A greenhouse experiment was conducted in which a variety of apple rootstock genotypes (G.890, G.935, M.26, and M.7) were inoculated with a commercially available, multi-species AMF consortium. Nursery-derived AMF communities were sequenced, and changes to AMF community structure following cultivation in pasteurized soil (inoculated and non-inoculated) were assessed using a Glomeromycota-specific phylogenetic tree, which included 91 different AMF species from 24 genera. Results show that inoculant colonization potential was limited and that apple rootstocks serve as a significant source of inoculum from the nursery where they are produced. Rootstocks established relationships with introduced AMF in a genotype-specific manner. Regardless of colonization success, however, the inoculant caused alterations to the resident AMF communities of both Geneva and Malling rootstocks, particularly low abundance taxa. In addition, phylogeny-based analysis revealed a unique, well-supported clade of unknown taxonomy, highlighting the importance of using phylogenetic-based classification for accurate characterization of AMF communities.IMPORTANCEUnderstanding the impacts of introduced AMF on residential AMF communities is essential to improving plant productivity in nursery and orchard systems. In general, there is a dearth of data on the interactions of commercial AMF inoculants with pre-established AMF communities living in symbiosis with the host plant. The interplay between apple rootstock genotype and the endophytic root microbiome is also an area where more research is needed. This study demonstrates the potential for nursery-established AMF associations to be maintained when transplanted into the field. In addition to providing insight into rootstock/AMF associations, our study calls attention to the current issues attendant with relying on web-based databases for determining AMF identity. The use of phylogenetic tools represents one possible solution and may be of value to industry practitioners in terms of improving product composition and consistency.

Clostridioides difficile is an obligate anaerobic, Gram-positive bacterium that produces toxins. Despite technological progress, conducting gene expression analysis of C. difficile under different conditions continues to be labor-intensive. Therefore, there is a demand for simplified tools to investigate the transcriptional and translational regulation of C. difficile. The cell-free gene expression (CFE) system has demonstrated utility in various applications, including prototyping, protein production, and in vitro screening. In this study, we developed a C. difficile CFE system capable of in vitro transcription and translation (TX-TL) in the presence of oxygen. Through optimization of cell extract preparation and reaction systems, we increased the protein yield significantly. Furthermore, our observations indicated that this system exhibited higher protein yield using linear DNA templates than circular plasmids for in vitro expression. The prototyping capability of the C. difficile CFE system was assessed using a series of synthetic Clostridium promoters, demonstrating a good correlation between in vivo and in vitro expression. Additionally, we tested the expression of tcdB and tcdR from clinically relevant C. difficile strains using the CFE system, confirming higher toxin expression of the hypervirulent strain R20291. We believe that the CFE system can not only serve as a platform for in vitro protein synthesis and genetic part prototyping but also has the potential to be a simplified model for studying metabolic regulations in Clostridioides difficile.IMPORTANCEClostridioides difficile has been listed as an urgent threat due to its antibiotic resistance, and it is crucial to conduct gene expression analysis to understand gene functionality. However, this task can be challenging, given the need to maintain the bacterium in an anaerobic environment and the inefficiency of introducing genetic material into C. difficile cells. Conversely, the C. difficile cell-free gene expression (CFE) system enables in vitro transcription and translation in the presence of oxygen within just half an hour. Furthermore, the composition of the CFE system is adaptable, permitting the addition or removal of elements, regulatory proteins for example, during the reaction. As a result, this system could potentially offer an efficient and accessible approach to accelerate the study of gene expression and function in Clostridioides difficile.

Trueperella pyogenes is an important bacterial pathogen implicated in infections such as mastitis, metritis, pneumonia, and liver abscesses in both domestic and wild animals, as well as endocarditis and prosthetic joint infections in humans. Understanding the genomic and metabolic features that enable T. pyogenes to colonize different anatomical sites within a host and its inter-kingdom transmission and survival is important for the effective control of this pathogen. We employed whole-genome sequencing, phenotype microarrays, and antimicrobial susceptibility testing to identify genomic, metabolic and phenotypic features, and antimicrobial resistance (AMR) genes in T. pyogenes recovered from different livestock, companion, and wildlife animals. For comparative genomic analysis, 83 T. pyogenes genomes, including 60 isolated in the current study and 23 publicly available genomes were evaluated. These genomes represented T. pyogenes strains originating from 16 different body sites of 11 different animal hosts (e.g., cattle, swine, ovine, deer, bison, horse, chamois, and cat). Additionally, 49 T. pyogenes isolates (cattle, sheep, deer, swine, and cats) were evaluated for phenotypic AMR using disk diffusion, and for metabolic profiling using the Biology GENIII MicroPlates. The T. pyogenes strains were found not to be host- or body site-specific. The presence of conserved virulence genes (plo and fimA), as well as genotypic and phenotypic AMR may contribute to the ability of T. pyogenes to cause infections in livestock, wildlife, and pets. Most of the tested isolates metabolized diverse carbon sources and chemical compounds, suggesting that this metabolic versatility may enhance the survival, competitiveness, and pathogenic potential of T. pyogenes.IMPORTANCETrueperella pyogenes is an important animal pathogen with zoonotic potential, posing a significant health concern to both animals and humans due to its ability to cause infections across different animal host species and tissues. Current understanding of this pathogen's adaptability and survival mechanisms is limited. Here, we evaluated the genomic, virulence, metabolic, and antimicrobial resistance (AMR) characteristics of T. pyogenes recovered from 16 different body sites of 11 different animal hosts (livestock, companion, and wild animals). We identified multiple AMR and virulence genes that may enable T. pyogenes for sustained infection and transmission. Additionally, T. pyogenes strains displayed metabolic versatility which could also contribute to its ability to thrive in diverse environments. Understanding the genomic and metabolic, and AMR characteristics that enable T. pyogenes to colonize different anatomical sites within a host and its transmission between different animal species is important for the effective control of this pathogen.

There is growing interest in members of the genus Segatella (family Prevotellaceae) as members of a well-balanced human gut microbiota (HGM). Segatella are particularly associated with the consumption of a diet rich in plant polysaccharides comprising dietary fiber. However, understanding of the molecular basis of complex carbohydrate utilization in Segatella species is currently incomplete. Here, we used RNA sequencing (RNA-seq) of the type strain Segatella copri DSM 18205 (previously Prevotella copri CB7) to define precisely individual polysaccharide utilization loci (PULs) and associated carbohydrate-active enzymes (CAZymes) that are implicated in the catabolism of common fruit, vegetable, and grain polysaccharides (viz. mixed-linkage β-glucans, xyloglucans, xylans, pectins, and inulin). Although many commonalities were observed, several of these systems exhibited significant compositional and organizational differences vis-à-vis homologs in the better-studied Bacteroides (sister family Bacteroidaceae), which predominate in post-industrial HGM. Growth on β-mannans, β(1, 3)-galactans, and microbial β(1, 3)-glucans was not observed, due to an apparent lack of cognate PULs. Most notably, S. copri is unable to grow on starch, due to an incomplete starch utilization system (Sus). Subsequent transcriptional profiling of bellwether Ton-B-dependent transporter-encoding genes revealed that PUL upregulation is rapid and general upon transfer from glucose to plant polysaccharides, reflective of de-repression enabling substrate sensing. Distinct from previous observations of Bacteroides species, we were unable to observe clearly delineated substrate prioritization on a polysaccharide mixture designed to mimic in vitro diverse plant cell wall digesta. Finally, co-culture experiments generally indicated stable co-existence and lack of exclusive competition between S. copri and representative HGM Bacteroides species (Bacteroides thetaiotaomicron and Bacteroides ovatus) on individual polysaccharides, except in cases where corresponding PULs were obviously lacking.

Importance: There is currently a great level of interest in improving the composition and function of the human gut microbiota (HGM) to improve health. The bacterium Segatella copri is prevalent in people who eat plant-rich diets and is therefore associated with a healthy lifestyle. On one hand, our study reveals the specific molecular systems that enable S. copri to proliferate on individual plant polysaccharides. On the other, a growing body of data suggests that the inability of S. copri to grow on starch and animal glycans, which dominate in post-industrial diets, as well as host mucin, contributes strongly to its displacement from the HGM by Bacteroides species, in the absence of direct antagonism.

Agricultural water is a potential source of microbial contamination whereby Escherichia coli, Salmonella, Cryptosporidium, and Cyclospora cayetenensis can enter the food supply. To reduce this risk, effective sanitization of agricultural water may be critical to food safety. As such, it is important to investigate the effects of aqueous peracetic acid (PAA) and chlorine (Cl) on bacteria and protozoa at different treatment times and temperatures in agricultural water with respect to key water characteristics. Multiple concentrations of each sanitizer, ranging from 3 to 200 ppm, were prepared in recently collected agricultural water, the solution was brought to the desired temperature, and the target organisms were added and left for the desired contact time (5 or 10 minutes) when sodium metabisulfite was added to neutralize the sanitizers. Bacterial samples were enumerated on MacConkey or XLT4 agar. Samples with protozoa were added to mammalian cell culture (HCT-8 cells for Cryptosporidium parvum and MDBK cells for Eimeria tenella). After 48 hours, the infected cells were collected, DNA extracted and infectivity assessed by quantitative PCR (qPCR). Low and high concentrations of sanitizer were effective at eliminating bacteria with Cl being significantly (P < 0.05) more effective. The greatest reductions in E. coli and Salmonella (3.48 log and 2.5 log cfu/mL, respectively) were observed after 10 minutes of exposure to 10 ppm Cl. Concentrations of sanitizer 50 ppm and lower resulted in insignificant (P > 0.05) reductions in parasite infectivity of less than 1 log for both organisms. A 200 ppm PAA treatment reduced infectious oocyst populations by 3.8 log for C. parvum and 2.6 log for E. tenella, with Cl being significantly (P < 0.05) less effective against these organisms.

Importance: This research is critical to inform decisions regarding the application and use of sanitizers in pre-harvest agricultural water settings to enhance food safety. Understanding the effectiveness of chlorine (Cl) and peracetic acid (PAA) on bacteria and protozoa will allow for the more efficient and practical use of these sanitizers, thus improving agricultural practices in ways that are beneficial to both growers and consumers.

Deinococcus radiodurans, a natural transformation (NT)-enabled bacterium renowned for its exceptional radiation resistance, employs unique DNA repair and oxidative stress mitigation mechanisms as a strategic response to DNA damage. This study excavates into the intricate roles of NT machinery in the stressed D. radiodurans, focusing on the genes comEA, comEC, endA, pilT, and dprA, which are instrumental in the uptake and processing of extracellular DNA (eDNA). Our data reveal that NT not only supports the nutritional needs of D. radiodurans under stress but also has roles in DNA repair. The study findings establish that NT-specific proteins (ComEA, ComEC, and endonuclease A [EndA]) may contribute to support the nutritional requirements in unstressed and heavily DNA-damaged cells, while DprA contributes differently and in a context-dependent manner to navigating through the DNA damage storm. Thus, this dual functionality of NT-specific genes is proposed to be a contributing factor in the remarkable ability of D. radiodurans to survive and thrive in environments characterized by high levels of DNA-damaging agents.IMPORTANCEDeinococcus radiodurans is a bacterium known for its extraordinary radiation resistance. This study explores the roles of NT machinery in the radiation-resistant bacterium Deinococcus radiodurans, focusing on the genes comEA, comEC, endA, pilT, and dprA. These genes are crucial for the uptake and processing of eDNA and contribute to the bacterium nutritional needs and DNA repair under stress. The findings suggest that the NT-specific proteins ComEA, ComEC, and EndA may help meet the nutritional needs of unstressed and heavily DNA-damaged cells, whereas DprA plays a distinct role that varies, depending on the context in aiding cells to cope with DNA damage. The functionality of NT genes is proposed to enhance D. radiodurans survival in environments with high levels of DNA-damaging agents.

The cofactor regeneration system plays a crucial role in redox biocatalysis for organic synthesis and the pharmaceutical industry. The alcohol dehydrogenase (ADH)-based regeneration system offers a promising solution for the in situ regeneration of NAD(P)H. However, its widespread use is hindered by low activity and poor expression of ADH in Escherichia coli. Herein, the BioBricks (promoter, ribosome binding site [RBS], functional gene, and terminator) were assembled and engineered to constitute an efficient NADH regeneration system. The semi-rational design was employed to enhance the catalytic efficiency of GstADH (an ADH from Geobacillus stearothermophilus), resulting in a beneficial GstADH variant with a 2.1-fold increase in catalytic efficiency. Furthermore, the RBS optimization was used to increase the expression of ADH genes, leading to the identification of an RBS with a 3.2-fold increased translation rate. Using this developed system, the NADH generating velocity reached more than 2 s^-1 even toward 0.1 mM NAD⁺, indicating that it is the most promising NADH regeneration so far. Finally, the engineered system was utilized for the asymmetric biosynthesis of l-phosphinothricin (a chiral herbicide), with a high yield (>95%).

Importance: The alcohol dehydrogenase (ADH)-based coenzyme regeneration system serves as a useful tool in redox biocatalysis. This system effectively replenishes NAD(P)H by utilizing isopropanol as a substrate, with the added advantage of easily separable acetone as a by-product. Previous studies focused on discovering new adh genes and engineering the ADH protein for higher catalytic efficiency, neglecting the optimization of other gene components. In this study, a remarkably efficient NADH regeneration system was developed using BioBricks assembly for system initialization. The ADH engineering was used to enhance catalytic efficiency, and RBS optimization for elevated ADH expression, which resulted in not only a 2.1-fold increase in catalytic efficiency but also a 3.2-fold increase in translation rate. Together, these improvements resulted in an overall 6.7-fold enhancement in performance. This system finds application in a wide range of NADH-dependent biocatalysis processes and is particularly advantageous for the biosynthesis of fine chemicals.

Electroactive organisms contribute to metal cycling, pollutant removal, and other redox-driven environmental processes via extracellular electron transfer (EET). Unfortunately, developing genotype-phenotype relationships for electroactive organisms is challenging because EET is necessarily removed from the cell of origin. Microdroplet emulsions, which encapsulate individual cells in aqueous droplets, have been used to study a variety of extracellular phenotypes but have not been applied to investigate EET. Here, we describe the development of a microdroplet emulsion system to sort and enrich EET-capable organisms from complex populations. We validated our system using the model electrogen Shewanella oneidensis and described the tooling of a benchtop microfluidic system for oxygen-limited conditions. We demonstrated the enrichment of strains exhibiting electroactive phenotypes from mixed wild-type and EET-deficient populations. As a proof-of-concept application, we collected samples from iron sedimentation in Town Lake (Austin, TX) and subjected them to microdroplet enrichment. We measured an increase in electroactive organisms in the sorted population that was distinct compared to a population growing in bulk culture with Fe(III) as the sole electron acceptor. Finally, two bacterial species not previously shown to be EET-capable, Cronobacter sakazakii and Vagococcus fessus, were further cultured and characterized for electroactivity. Our results demonstrate the utility of microdroplet emulsions for isolating and identifying EET-capable bacteria.IMPORTANCEThis work outlines a new high-throughput method for identifying electroactive bacteria from mixed populations. Electroactive bacteria play key roles in iron trafficking, soil remediation, and pollutant degradation. Many existing methods for identifying electroactive bacteria are coupled to microbial growth and fitness-as a result, the contributions from weak or poor-growing electrogens are often muted. However, extracellular electron transfer (EET) has historically been difficult to study in high-throughput in a mixed population since extracellular reduction is challenging to trace back to the parent cell and there are no suitable fluorescent readouts for EET. Our method circumvents these challenges by utilizing an aqueous microdroplet emulsion wherein a single cell is statistically isolated in a pico- to nano-liter-sized droplet. Then, via fluorescence obtained from copper reduction, the mixed population can be fluorescently sorted and gated by performance. Utilizing our technique, we characterize two previously unrecognized weak electrogens Vagococcus fessus and Cronobacter sakazakii.