Applied and Environmental Microbiology

An air sanitizer was evaluated using an aerobiology protocol, compliant with the U.S. Environmental Protection Agency’s Air Sanitizer Guidelines, for virucidal activity against bacteriophages Phi6 and MS2 (used as surrogates for enveloped and non-enveloped human pathogenic viruses). The phages were suspended in a medium containing a tripartite soil load simulating body fluids and aerosolized using a six-jet Collison nebulizer in an enclosed 25 m ³ aerobiology chamber at 22 ± 2°C and 50 ± 10% relative humidity. The air sanitizer was sprayed into the chamber for 30 s. Viable phages in the air were captured directly, in real time, on host bacterial lawns using a slit-to-agar sampler. Reductions in viable phage concentration ≥3.0 log 10 (99.9%) were observed after a mean exposure of 3.6 min for Phi6, suggesting efficacy against enveloped viruses (e.g., SARS-CoV-2, influenza, and RSV), and ~10.6 min for MS2, suggesting virucidal efficacy for non-enveloped viruses (e.g., noroviruses and rhinoviruses). This targeted air sanitization approach represents an important non-pharmaceutical public health intervention with virucidal efficacy against airborne viral pathogens. IMPORTANCE Airborne viruses are implicated in the transmission indoors of respiratory and enteric viral infections. Air sanitizers represent a non-pharmaceutical intervention to mitigate the risk of such viral transmission. We have developed a method that is now an ASTM International standard (ASTM E3273-21) as well as a test protocol approved by the U.S. EPA to evaluate the efficacy of air sanitizing sprays for inactivating airborne MS2 and Phi6 bacteriophage (used as surrogates for non-enveloped and enveloped human pathogenic viruses, respectively). The test phages were individually suspended in a soil load and aerosolized into a room-sized aerobiology chamber maintained at ambient temperature and relative humidity. Reductions in viable phage concentration ≥3.0 log 10 (99.9%) were observed after a mean exposure of 3.6 min for Phi6, suggesting efficacy against enveloped viruses (e.g., SARS-CoV-2; influenza; RSV), and ~10.6 min for MS2, suggesting virucidal efficacy for non-enveloped viruses (e.g., noroviruses and rhinoviruses). The data suggest the utility of the air sanitizer for mitigating the risk of indoor viral transmission during viral pandemics and outbreaks.

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.

Microbial nitrate reduction coupled to iron(II) oxidation (NRFeOx) occurs in paddy soils due to high levels of dissolved iron(II) and regular application of nitrogen fertilizer. However, to date, there is no lithoautotrophic NRFeOx isolate or enrichment culture available from this soil environment. Thus, resulting impacts on greenhouse gas emissions during nitrate reduction (i.e., nitrous oxide [N 2 O]) and on toxic metalloid (i.e., arsenic) mobility can hardly be investigated. We enriched a lithoautotrophic NRFeOx culture, culture HP (Huilongpu paddy, named after its origin), from a paddy soil (Huilongpu Town, China), which was dominated by Gallionella (71%). The culture reduced 0.45 to 0.63 mM nitrate and oxidized 1.76 to 2.31 mM iron(II) within 4 days leading to N 2 O as the main N-product (62%–88% N 2 O-N of total reduced NO 3 ⁻ -N). Nitrite was present as an intermediate at a maximum of 0.16 ± 0.1 mM. Cells were associated with, but mostly not encrusted by, poorly crystalline iron(III) minerals (ferrihydrite). Culture HP performed best below an iron(II) threshold of 2.5–3.5 mM and in a pH range of 6.50–7.05. In the presence of 100 µM arsenite, only 0%–18% of iron(II) was oxidized. Due to low iron(II) oxidation, arsenite was not immobilized. However, the proportion of N 2 O-N of total reduced NO 3 ⁻ -N decreased from 77% to 30%. Our results indicate that lithoautotrophic NRFeOx occurs even in organic-rich paddy soils, resulting in denitrification and subsequent N 2 O emissions. The obtained novel enrichment culture allows us to study the impact of lithoautotrophic NRFeOx on arsenic mobility and N 2 O emissions in paddy soils. IMPORTANCE Paddy soils are naturally rich in iron(II) and regularly experience nitrogen inputs due to fertilization. Nitrogen fertilization increases nitrous oxide emissions as it is an intermediate product during nitrate reduction. Microorganisms can live using nitrate and iron(II) as electron acceptor and donor, respectively, but mostly require an organic co-substrate. By contrast, microorganisms that only rely on nitrate, iron(II), and CO 2 could inhabit carbon-limited ecological niches. So far, no isolate or consortium of lithoautotrophic iron(II)-oxidizing, nitrate-reducing microorganisms has been obtained from paddy soil. Here, we describe a lithoautotrophic enrichment culture, dominated by a typical iron(II)-oxidizer ( Gallionella ), that oxidized iron(II) and reduced nitrate to nitrous oxide, negatively impacting greenhouse gas dynamics. High arsenic concentrations were toxic to the culture but decreased the proportion of nitrous oxide of the total reduced nitrate. Our results suggest that autotrophic nitrate reduction coupled with iron(II) oxidation is a relevant, previously overlooked process in paddy soils.

Wood-fall micro-ecosystems contribute to biogeochemical processes in the oligotrophic deep ocean. However, the community assembly processes and biogeochemical functions of microbiomes in wood fall remain unclear. This study investigated the diversity, community structure, assembly processes, and functional profiles of bacteria and fungi in a deep-sea wood fall from the South China Sea using physicochemical indices, amplicon sequencing, and metagenomics. The results showed that distinct wood-fall contact surfaces exhibit habitat heterogeneity. The bacterial community of all contact surfaces and the fungal community of seawater contact surface (SWCS) were affected by homogeneous selection. In SWCS and transition region (TR), bacterial communities were influenced by dispersal limitation, whereas fungal communities were affected by homogenizing dispersal. The Venn diagram visualization revealed that the shared fungal community between SWCS and TR was dominated by Aspergillaceae. Additionally, the bacterial community demonstrated a higher genetic potential for sulfur, nitrogen, and methane metabolism than fungi. The sediment contact surface enriched modules were associated with dissimilatory sulfate reduction and methanogenesis, whereas the modules related to nitrate reduction exhibited enrichment characteristics in TR. Moreover, fungi showed a stronger potential for lignocellulase production compared to bacteria, with Microascaceae and Nectriaceae identified as potential contributors to lignocellulose degradation. These results indicate that environmental filtering and organism exchange levels regulated the microbial community assembly of wood fall. The biogeochemical cycling of sulfur, nitrogen, and methane was mainly driven by the bacterial community. Nevertheless, the terrestrial fungi Microascaceae and Nectriaceae might degrade lignocellulose via the combined action of multiple lignocellulases. IMPORTANCE The presence and activity of microbial communities may play a crucial role in the biogeochemical cycle of deep-sea wood-fall micro-ecosystems. Previous studies on wood falls have focused on the microbiome diversity, community composition, and environmental impact, while few have investigated wood-fall micro-ecosystems by distinguishing among distinct contact surfaces. Our study investigated the microbiome dynamics and functional profiles of bacteria and fungi among distinct wood-fall contact surfaces. We found that the microbiome community assembly was regulated by environmental filtering and organism exchange levels. Bacteria drive the biogeochemical cycling of sulfur, nitrogen, and methane in wood fall through diverse metabolic pathways, whereas fungi are crucial for lignocellulose degradation. Ultimately, this study provides new insights into the driving pattern of community assembly, biogeochemical processes, and lignocellulose degradation in the microbiomes of deep-sea wood-fall micro-ecosystems, enhancing our comprehension of the ecological impacts of organic falls on deep-sea oligotrophic environments.

Viral detection methodologies used for wastewater-based epidemiology (WBE) studies have a broad range of efficacies. The complex matrix and low viral particle load in wastewater emphasize the importance of the concentration method. This study focused on comparing three commonly used virus concentration methods: polyethylene glycol precipitation (PEG), immuno-magnetic nanoparticles (IMNP), and electronegative membrane filtration (EMF). Influent and effluent wastewater samples were processed by the methods and analyzed by DNA/RNA quantification and sequencing for the detection of human viruses. SARS-COV-2, Astrovirus, and Hepatitis C virus were detected by all the methods in both sample types. PEG precipitation resulted in the detection of 20 types of viruses in influent and 16 types in effluent samples. The corresponding number of virus types detected was 21 and 11 for IMNP, and 16 and 8 for EMF. Certain viruses were unique to only one concentration method. For example, PEG detected three types of viruses in influent and six types in effluent compared to IMNP, which detected seven types in influent and one type in effluent samples. However, the EMF method appeared to be the least effective, detecting three types in influent and none in effluent samples. Rotavirus was detected in influent sample using IMNP method, whereas EMF and PEG methods failed to yield a similar outcome. Consequently, the potential false negative results pose a risk to the credibility of WBE applications. Therefore, implementation of a proper concentration technique is critical to minimize method biases and ensure accurate viral profiling in WBE studies. IMPORTANCE In recent years, significant research efforts have been focused on the development of viral detection methodology for wastewater-based epidemiology studies, showing a range of variability in detection efficacies. A proper methodology is essential for an appropriate evaluation of disease prevalence and community health in such studies and necessitates designing a concentration method based on the target pathogenic virus. There remains a need for comparative performance evaluations of methods in the context of detection efficiencies. This study highlights the significant impact of sample matrix, viral structure, and nucleic acid composition on the efficacy of viral concentration methods. Assessing WBE techniques to ensure accurate detection and understanding of viral presence within wastewater samples is critical for revealing viral profiles in municipality wastewater samples.

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.

Cadmium (Cd) and chromium (Cr) are frequently encountered toxicants, while iron (Fe) plays a crucial role in bacterial survival under conditions of heavy metal stress. However, intracellular Fe ion depletion by heavy metals leads to a state of Fe starvation. Therefore, it is imperative to investigate the mechanism through which bacteria maintain a balance between heavy metal detoxification and Fe homeostasis. This study demonstrates Cd(II) immobilization and Cr(VI) reduction abilities of Stenotrophomonas sp. SY1, while proteomics reveals the upregulation of heme metabolism in response to Cd(II) and Cr(VI) exposure. The expression of the heme-uptake system in Escherichia coli can enhance Cd(II) immobilization and facilitate Cr(VI) reduction. The ferruginous hemeprotein HhuH exhibits the ability to chelate Cd(II) and reduce Cr(VI). The presence of Cd(II) and Cr(VI) in strain SY1 initially led to Fe starvation. Subsequently, the hemeprotein HhuH facilitated Cd(II) adsorption and Cr(VI) reduction, thereby restoring normal cellular Fe homeostasis. Our findings explain the hemeprotein-mediated mechanism for Cd(II) adsorption and Cr(VI) reduction, providing further insights into the correlation between heavy metal and Fe metabolism. IMPORTANCE Iron (Fe) is an indispensable trace element for many organisms, and virtually, all bacteria require Fe as a cofactor in enzymes to facilitate redox reactions involved in fundamental cellular processes during periods of heavy metal stress. Understanding bacterial response to Fe in heavy metal contamination is essential. Therefore, our study elucidates Cd(II) adsorption and Cr(VI) reduction processes mediated by the Fe-bearing hemeprotein HhuH. It is a unique trifunctional protein capable of chelating Cd(II) and reducing Cr(VI), demonstrating significant potential in the environmental remediation of heavy metals.

Natural chondroitin sulfate (CS), extracted from animal cartilage, is widely used in the pharmaceuticals and foods. However, contamination with animal-derived heteropolysaccharides presents significant challenges, including potential immune responses. To address this, we developed a green and efficient method for synthesizing chondroitin sulfate E (CSE) via enzymatic synthesis, identifying Ec CHST15, a sulfotransferase that catalyzes the conversion of chondroitin sulfate A (CSA) to CSE. We investigated the novel catalytic mechanism of CHST15 through quantum mechanical (QM) calculations and experimental validation, confirming its alignment with the SN2 reaction mechanism. Subsequently, we enhanced the catalytic efficiency of CHST15 using protein engineering, improving the catalytic efficiency from 18.1% in the wild type (WT) to 62.5% in the M7 mutant—a 3.5-fold increase. Finally, we constructed a six-enzyme cascade whole-cell catalyst, achieving a 72.2% conversion of 15 g/L CSA to produce CSE within 24 h. These findings offer a promising strategy for the industrial production of CSE. IMPORTANCE Current methods for obtaining chondroitin sulfate (CS) primarily rely on tissue extraction and chemical synthesis. However, these approaches are hindered by contamination risks from animal-derived heteropolysaccharides and the technical challenges inherent in complex chemical synthesis, limiting the scalability of industrial CS production. To address this, we developed a green and efficient enzymatic biosynthesis method for chondroitin sulfate E (CSE). By identifying and engineering the sulfotransferase CHST15 from Erpetoichthys calabaricus , we created a mutant ( Ec CHST15 M7 ) with a 3.5-fold increase in catalytic efficiency toward chondroitin sulfate A (CSA) compared to the wild-type enzyme. Additionally, we constructed a six-enzyme cascade whole-cell biocatalyst, achieving a 72.2% conversion rate from CSA to CSE. This study opens new avenues for the industrial-scale production of CSE through sustainable enzymatic processes.

Aedes aegypti mosquitoes are well adapted to dry climates and can retain their eggs for extended periods in the absence of suitable habitat. Wolbachia strains transferred from other insects to mosquitoes can be released to combat dengue transmission by blocking virus replication and spreading through populations, but host fitness costs imposed by Wolbachia , particularly under some environments, can impede spread. We, therefore, assessed the impact of two Wolbachia strains being released for dengue control ( w AlbB and w MelM) on fecundity and egg viability following extended egg retention (up to 24 days) under laboratory conditions. Egg viability following retention decreased to a greater extent in females carrying w MelM compared to uninfected or w AlbB females. Fertility fully recovered in uninfected females following a second blood meal after laying retained eggs, while w MelM females experienced only partial recovery. Effects of w MelM on egg retention were similar regardless of whether females were crossed to uninfected or w MelM males, suggesting that fitness costs were triggered by Wolbachia presence in females. The fecundity and hatch proportions of eggs of w MelM females declined with age, regardless of whether females used stored sperm or were recently inseminated. Costs of some Wolbachia strains during egg retention may affect the invasion and persistence of Wolbachia in release sites where larval habitats are scarce and/or intermittent. IMPORTANCE Wolbachia mosquito releases are expanding around the world with substantial impacts on dengue transmission. Releases have succeeded in many locations, but the establishment of Wolbachia has been challenging in some environments, and the factors contributing to this outcome remain unresolved. Here, we explore the effects of Wolbachia on a novel trait, egg retention, which is likely to be important for the persistence of mosquito populations in locations with intermittent rainfall. We find substantial impacts of the Wolbachia strain w MelM on the quality of retained eggs but not the w AlbB strain. This cost is driven by the Wolbachia infection status of the female and can partially recover following a second blood meal. The results of our study may help to explain the difficulty in establishing Wolbachia strains at some field release sites and emphasize the need to characterize Wolbachia phenotypes across a variety of traits and strains.

Wohlfahrtiimonas chitiniclastica is an emerging zoonotic pathogen associated with bacteremia, myiasis, and soft tissue infections. It is insufficiently identified and underestimated due to reasons, such as shortcomings of the traditional identification techniques and language barriers in local case reports from different regions. In this review, we summarize the currently available literature. In particular, we added previously overlooked cases from Chinese and other medical communities. The clinical characteristics, identification, and treatment of W. chitiniclastica are discussed. This work provides a complete review of the previous work including cases from human, animal, and other sources.

Diarrheal diseases attributable to multidrug-resistant F4+ enterotoxigenic Escherichia coli (ETEC) are escalating in severity, posing significant risks to the health and safety of both humans and animals. This study used Saccharomyces cerevisiae EBY100 to display the FaeG subunit of F4 colonizing factor as an oral vaccine against F4+ ETEC infection. Mice were orally immunized twice with 10 ⁸ CFU of EBY100/pYD1-FaeG, followed by a challenge with F4+ ETEC EC6 on day 7 post-immunization. The results showed that the recombinant strain EBY100/pYD1-FaeG orally enhanced the growth of the small intestine villi, significantly boosted the expression of tight junction proteins ( ZO-1 , Occludin , MUC2 , and Claudin ) ( P < 0.05), and modulated the gut microbiota composition. Additionally, immunization with EBY100/pYD1-FaeG also upregulated the levels of IL-2, IL-4, and IFN-γ in the intestines of mice ( P < 0.01), while serum IgG and fecal sIgA titer significantly increased ( P < 0.05). These immune responses enhanced the capacity to fight against ETEC, leading to an increased survival rate of mice and relieved damage to tissues and organs of mice infection. In summary, the study suggested that the recombinant Saccharomyces cerevisiae EBY100/pYD1-FaeG could effectively stimulate the immune response and generate specific antibodies against F4+ ETEC, showing its potential to serve as a subunit oral vaccine candidate for preventing F4+ ETEC infection. IMPORTANCE The multidrug-resistant F4+ enterotoxigenic Escherichia coli (ETEC) strains are the primary clinical pathogens responsible for post-weaning diarrhea in piglets, resulting in substantial economic losses in the pig farming industry. In the study, we developed an oral vaccine candidate, Saccharomyces cerevisiae EBY100/pYD1-FaeG, to prevent diarrhea caused by multidrug-resistant F4+ ETEC. Oral administration of EBY100/pYD1-FaeG significantly enhanced immune responses, improved intestinal health, and provided protection against F4+ ETEC infection in mice. This approach offers a potential application prospect for preventing F4+ ETEC infections that lead to post-weaning diarrhea in clinical settings and provides a promising solution for addressing the growing threat of antibiotic resistance in bacterial pathogens.

Karst tiankeng is a typical terrestrial habitat island-like system, known as an oasis in a degraded karst landscape. However, we know little about the composition, structure, and life strategies of soil microbial communities in the karst tiankeng ecosystem. In this study, we use amplicon sequencing to investigate the soil bacteria and fungi of 26 karst tiankeng in two typical karst tiankeng groups. The results showed that the composition and structure of bacterial and fungal communities were significantly different at two dimensions (among and within the karst tiankeng group). Bacteria showed more sensitivity to variation in the karst tiankeng area and isolation than fungi. With the increase of karst tiankeng area and isolation, the bacterial life strategies shift from K -strategist to r -strategist, likely due to the changes in soil properties (total phosphorus, Ca, and soil water content). Abundant and rare taxa play different roles in karst tiankeng ecosystems; abundant taxa serve a key role in nutrient cycles and life strategy shifts by occupying the key status in networks. Considering the key role of soil microbes in ecosystems, more attention must be paid to the impact of habitat loss on soil microbial life strategies, particularly in the ecological impact of life strategies change of abundant and rare taxa. IMPORTANCE These findings highlight that habitat loss or fragmentation induces a shift in microbial life strategies and improves our understanding of the composition and biogeography of karst ecosystem microorganisms.

Difficulties exist in obtaining full-length, correctly folded, and soluble papain or papain-like proteases that necessitate the exploration of alternative strategies. This study describes the development of an Escherichia coli strain capable of producing soluble papain without the need for complex and time-consuming in vitro refolding steps. To enhance the production of soluble papain, engineered T7 promoters and a recombinant papain translationally fused with varying tags were constructed. The tags investigated include the maltose-binding protein, small ubiquitin modifier protein, and glutathione transferase. An E. coli SHuffle strain was engineered to accumulate hydrogen peroxide (H 2 O 2 ) by disruption of the redox pathway. This was accomplished by co-expression of the fusion constructs with two human endoplasmic reticulum-resident proteins, thiol peroxidase glutathione peroxidase-7 (GPx7), and protein disulfide isomerase (PDI). The oxidizing capacity of H 2 O 2 was used to improve disulfide bond formation in papain. The GPx7-PDI fusion dyad played a significant role in consuming harmful H 2 O 2 generated by the SHuffle cells. This consumption of H 2 O 2 helped provide the necessary oxidizing conditions for the efficient production of soluble papain. In shake-flask experiments, the recombinant strain produced ~110 mg/L of papain. Moreover, in batch fermentation, the volumetric yield reached ~349 mg/L. This work provides insights into recombinant papain microbial production that can lead to an industrial viable production strain. IMPORTANCE Papain, a cysteine-like protease, has extensive applications across various industries including food, chemical, pharmaceutical, drug, and polymer. However, the traditional isolation of papain from Carica papaya plants results in a complex mixture of proteases. Such protease mixtures result in an inability to understand which component enzyme contributed to substrate conversions. Concentrations of constituent enzymes likely differ based on the ripeness of the papaya fruit. Also, constituent enzymes from papaya differ in optimal activity as a function of temperature and pH. Thus, by using papain-like enzymes from papaya fruit, valuable information on component enzyme activity and specificity is lost. Numerous methods have been reported to purify papain and papain-like enzymes from the crude mixture. Often, methods involve at least three steps including column chromatography to separate five cysteine proteases. Such procedures represent tedious processes to manufacture the pure enzymes in Carica papaya extracts. The numerous uses of papain for industrial processes, as well as the probability that certain components of papain crude mixtures will be preferred for specific applications, necessitate alternative methods such as recombinant expression from microbial production systems to meet the high world demand for papain.

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 . IMPORTANCE 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. 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.

Previous work reported that deletion of the Enzyme IIAB subunits (EIIAB Man and manL ) of the glucose phosphotransferase system (PTS) (glucose-PTS, manLMNO ) in Streptococcus sanguinis impacted carbon catabolite repression and bacterial fitness. Here, a single-nucleotide polymorphism in ManN, ManNA91E, produced the unusual phenotype of increased excretion of organic acids and H 2 O 2 yet elevated PTS activities. To characterize the contributions of each component of the glucose-PTS to bacterial fitness, we performed genetic analyses by deleting from S. sanguinis SK36 the entire operon and each EII Man subunit individually; and genes encoding the catabolite control protein A (Δ ccpA ) and the redox regulator Rex (Δ rex ) for comparison. Deletion of each subunit incurred a growth defect on glucose partly due to elevated excretion of H 2 O 2 ; when supplemented with catalase, this defect was rescued, instead resulting in a significantly higher yield than the parent. All glucose-PTS deletion mutants presented an increased antagonism against the oral pathobiont Streptococcus mutans , a phenotype absent in Δ ccpA despite increased H 2 O 2 output. A shift in the pyruvate node toward mixed acid fermentation and increased arginine deiminase activity enhanced pH homeostasis in glucose-PTS mutants but not Δ ccpA . Despite the purported ability of Rex to regulate central carbon metabolism, deletion of rex had no significant impact on most of the phenotypes discussed here. These findings place glucose-PTS in the pivotal position of controlling central carbon flux in streptococci, with critical outcomes impacting acidogenicity, aciduricity, pH homeostasis, and antagonism, highlighting its potential as a therapeutic target for treating diseases with a dysbiotic microbiome. IMPORTANCE Management of carbohydrate metabolism and environmental stress is key to the survival of oral commensal species such as S. sanguinis . Antagonism of oral pathobionts and modulation of the environmental pH and oxidative potential by commensals are crucial to the maintenance of microbial homeostasis and prevention of oral diseases including dental caries. It is therefore vital to understand how these species regulate sugar fermentation, production of acids and ammonia, and stress management in an environment known for a feast-and-famine cycle of carbohydrates and similar fluctuations in pH and oxygen tension. Here, we detail that genetic alterations of the glucose-PTS transporter in S. sanguinis can significantly affect the regulation of factors required for bacterial fitness and homeostatic ability independent of known catabolic regulators. It is then discussed how these changes may impact the survival of streptococcal species and affect caries onset.

Postdocs are essential to microbial science and STEM academic workforces but are underpaid and receive little-to-no relocation benefits. PhDs foregoing postdoctoral training for lucrative industry and government jobs exit the academic pipeline, which imperils current scholarship and the future professoriate. Relocation to postdoc jobs is expensive, especially for recent graduates and international scholars, but academia rarely provides support. Solving this short-term liquidity pressure can increase productivity, job satisfaction, and the likelihood they remain in academia.

Increased tolerance to cadmium in Listeria monocytogenes has been suggested to contribute to their persistence in natural and food production environments. This study investigated the phenotypic cadmium response of L. monocytogenes strains with efflux pump cadAC (variants 1–4) and related strains with cadA1C1 . Growth of cadAC variant strains ( n = 5) in 0 µM–120 µM cadmium salts (CdCl 2 , CdSO 4 ) in Mueller-Hinton broth (MHB) was evaluated. Additionally, 88 L . monocytogenes strains from dairy processing facilities were exposed to 43.8 µM CdCl 2 in MHB, and their lag phase duration (LPD) was measured. Strains with cadA1 through cadA3 showed similar growth trends in the presence of cadmium, while the cadA4 variant (Scott A) had the highest CdCl 2 minimum inhibitory concentration (175 µM). Growth varied between the two salts, with CdSO 4 significantly increasing LPD ( P < 0.05) compared to CdCl 2 . In 43.8 µM CdCl 2 , cadA1 strains displayed LPDs ranging from 0.99 ± 0.14 h to 6.44 ± 0.08 h, with no clear genomic differences explaining this variability. Strains without cadA did not grow at 43.8 µM CdCl 2 but exhibited low tolerance (10.9 µM CdCl 2 ), potentially due to non-specific soft metal ATPases (626 aa; 737 aa) and soft metal resistance proteins encoded by czc genes (289 aa; 291 aa; 303 aa) within their chromosomes. These findings enhance our understanding of L. monocytogenes cadmium tolerance and underscore the need for further research to explore the genetic and physiological factors underlying these trends. IMPORTANCE Mobile genetic elements in Listeria monocytogenes contribute to its survival in natural and food processing environments. This study focused on how different genetic variants of the efflux pump gene cadAC and group of closely related cadA1C1 strains respond to cadmium exposure. When exposed to two cadmium salts, cadmium chloride and cadmium sulfate, we observed varying growth patterns, with a significantly longer lag phase in cadmium sulfate compared to cadmium chloride. Strains with cadA1 to cadA3 had similar growth trends, whereas a strain with the cadA4 variant had the highest minimum inhibitory concentration value. Among 88 strains from dairy processing facilities, significant phenotypic differences were observed despite core genome similarities, indicating other underlying genetic and physiological factors contribute to cadmium tolerance. Since cadmium tolerance studies in L. monocytogenes are limited, with rare phenotypic comparisons between closely related strains, our study makes an important observation and contribution to understanding of L. monocytogenes tolerance to cadmium by providing phenotypic comparisons between numerous strains within the same clonal group (<16 single nucleotide polymorphisms).

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.

The airborne transmission of infectious diseases and bioaerosol-induced cross-contamination pose significant challenges in the food, dairy, and pharma industries. This study evaluated the effectiveness of 279 nm UV-C LED irradiation for decontaminating bioaerosols, specifically containing microorganisms such as Escherichia coli (C3040- Kanamycin resistant), Salmonella Enteritidis (ATCC 4931), and Pseudomonas fragi (ATCC 4973), on food contact surfaces. Borosilicate glass, silicon rubber, and stainless steel (316L) surfaces were selected for experimentation for their usage in the food industry. A 50 µL cell suspension was aerosolized at 25 psi pressure using a 4-jet BLAM Nebulizer within a customized glass chamber and then deposited onto the surface of the coupons. The serial dilution approach was used for the microbial enumeration, followed by duplicate plating. With a low Root Mean Square Error (RMSE) and high R ² values, the biphasic kinetic model for UV-C inactivation curves of all three pathogens demonstrated the excellent goodness of fit parameters. At a UV-C dose of 6 mJ cm ⁻² , glass surfaces showed the maximum microbial inactivation (i.e., 2.80, 3.81, and 3.56 log CFU/mL for E. coli , Salmonella , and P. fragi , respectively). Stainless steel and silicon rubber surfaces showed significant microbial inactivation, but log 10 reductions observed were consistently lower than glass surface. Our research indicates that UV-C LEDs (279 nm) can effectively disinfect bioaerosols on food contact surfaces. IMPORTANCE Food safety is a major public health concern, with contaminated food causing serious illnesses. UV-C light, used for germicidal action, is effective in disinfecting surfaces and is not subject to the same strict legal restrictions as chemical disinfectants, simplifying compliance with food safety regulations. In this study, we evaluated the efficacy of UV-C (279 nm) LED systems for inactivation of surface-deposited bioaerosols of kanamycin-resistant Escherichia coli (C3040), Salmonella Enteritidis (ATCC 4931), and Pseudomonas fragi (ATCC 4973). The research outcomes can be used to develop UV-based surface disinfection systems to minimize the risk of foodborne illnesses and enhance safety in high-traffic food preparation areas.

Marine sponges are a prolific source of biologically active small molecules, many of which originate from sponge-associated bacteria. Identifying the producing bacteria is a key step in developing sustainable routes for the production of these metabolites. To facilitate the required computational analyses, we developed MetaSing, a reproducible singularity-based pipeline for assembly, identification of high-quality metagenome-assembled genomes (MAGs), and analysis of biosynthetic gene clusters (BGCs) from metagenomic short-read data. We applied this pipeline to metagenomic sequencing data from 16 marine sponges collected from New Zealand, Tonga, and the Mediterranean Sea. This analysis yielded 643 MAGs representing 510 species. Of the 2,670 BGCs identified across all samples, 70.8% were linked to a MAG. Comparison of BGCs to those identified from previously sequenced bacteria revealed high biosynthetic novelty in variety of underexplored phyla, including Poribacteria, Acidobacteriota, and Dadabacteria. Alongside the observation that each sample contains unique biosynthetic potential, this holds great promise for natural product discovery and for furthering the understanding of different sponge holobionts. IMPORTANCE Discovery of new chemical compounds such as natural products is a crucial endeavor to combat the increasing resistance to antibiotics and other drugs. This manuscript demonstrates that microbial communities associated with marine sponges investigated in this work encode the potential to produce novel chemistry. Lesser studied bacterial taxa that are often difficult to cultivate are particularly rich in potential.

As a response regulator of the EsrA-EsrB two-component system, EsrB is conserved in Hafniaceae and plays a crucial role in virulence and pathogenicity. EsrB possesses DNA binding abilities, enabling it to regulate the transcription of virulence genes to confront different stresses and achieve systematic infections. Here, ChIP-seq analysis of EsrB in Dulbecco’s Modified Eagle’s Medium (DMEM) (mimicking in vivo environments) revealed that EsrB preferred to bind to virulence-associated promoters with a distinct 7'-4-7'' pseudopalindromic DNA motif and interact with metabolic-related promoters with a high AT DNA motif. The crystal structure of the C-terminal of EsrB (EsrB C ) was solved at 2.20-Å resolution. Specifically, Lys ¹⁸¹ enabled the DNA-binding affinity of EsrB and promoted the in vitro and in vivo pathogenicity of Edwardsiella piscicida . Moreover, EsrB directly regulated the expression of genes associated with basal metabolism, including iron and tricarboxylic acid (TCA) cycles. Furthermore, EsrB enhanced iron transport capability and the enzyme activity of succinate dehydrogenase and pyruvate dehydrogenase in DMEM. Collectively, our structural and ChIP-seq analysis provides valuable insights into the DNA binding mechanism of EsrB which will facilitate our understanding of EsrB coordinating virulence and basal metabolism gene expression. IMPORTANCE As a crucial virulence regulator, EsrB possesses a LuxR-like superfamily domain at the C-terminal, which is conserved within the canonical NarL family regulators. Due to its critically important role in virulence and pathogenicity in fish hosts, the DNA binding ability has been believed to allow EsrB to regulate genes associated with the invasion process of host cells and basal metabolism in response to environmental stimuli. The lack of EsrB’s crystal structure has been a major obstacle in understanding the molecular mechanisms of EsrB-DNA interaction which choreographs EsrB-mediated pathogenic behavior. Here, we conducted ChIP-seq and solved the crystal structure of the C-terminal of EsrB (EsrBC) at 2.20-Å resolution, which revealed that EsrB preferred to bind to virulence-associated promoters with a distinct 7'-4-7' pseudopalindromic DNA motif and interacted with metabolic-related promoters with a high AT DNA motif in Dulbecco’s Modified Eagle’s Medium (DMEM) (mimicking in vivo environments). Our results facilitate a detailed understanding of EsrB’s regulatory role in Edwardsiella piscicida pathogenesis and expand our knowledge of virulence regulators in the family Hafniaceae .

Mammalian alkaline phosphatase (AP) is widely used in diagnostics and molecular biology but its widespread use is impaired because it is difficult to express in Escherichia coli and has low thermostability. To overcome these challenges, we employed sequence-based protein redesign methods, specifically full consensus design (FCD) and ancestral sequence reconstruction (ASR), to create APs with enhanced properties. Biochemical analyses revealed that these newly designed APs exhibited improved levels of expression in their active form and increased thermostability compared to bovine intestinal AP isozyme II (bIAPII), without impeding enzymatic activity. Notably, the activity in culture broth of the designed APs was an order of magnitude higher than that of bIAPII, and their thermal stability increased by 13°C–17°C (measured as T 50 ). We also assessed 28 single-point mutants of bIAPII to identify regions influencing thermostability and expression level; these mutations were common in the engineered APs but not in bIAPII. Specific mutations, such as T413E and G402S, enhanced thermostability, whereas increasing the expression level required multiple mutations. This suggests that a synergistic effect is required to enhance the expression level. Mutations enhancing thermostability were located in the crown domain, while those improving expression were close to the dimer interface, indicating distinct mechanisms underpinning these enhancements. IMPORTANCE Sequence-based protein redesign methods, such as full consensus design (FCD) and ancestral sequence reconstruction (ASR), have the potential to construct new enzymes utilizing protein sequence data registered in a rapidly expanding sequence database. The high thermostability of these enzymes would expand their application in diagnostics and molecular biology. These enzymes have also demonstrated a high level of active expression by Escherichia coli . These characteristics make these APs attractive candidates for industrial application. In addition, different amino acid residues are primarily responsible for thermal stability and active expression, suggesting important implications for strategies for designing enzymes by FCD and ASR.

The Atacama Desert in Chile is one of the driest and most inhospitable places on Earth. To analyze the diversity and distribution of microbial communities in such an environment, one of the most important and challenging steps is DNA extraction. Using commercial environmental DNA extraction protocols, a mixture of living, dormant, and dead cells of microorganisms is extracted, but separation of the different DNA pools is almost impossible. To overcome this problem, we applied a novel method on soils across a west–east moisture transect in the Atacama Desert to distinguish between extracellular DNA (eDNA) and intracellular DNA (iDNA) at the cell extraction level. Here, we show that a large number of living and potentially active microorganisms, such as Acidimicrobiia , Geodermatophilaceae , Frankiales , and Burkholderiaceae, occur in the hyperarid areas. We observed viable microorganisms involved as pioneers in initial soil formation processes, such as carbon and nitrogen fixation, as well as mineral-weathering processes. In response to various environmental stressors, microbes coexist as generalists or specialists in the desert soil environment. Our results show that specialists compete in a limited range of niches, while generalists tolerate a wider range of environmental conditions. Use of the DNA separation approach can provide new insights into different roles within viable microbial communities, especially in low-biomass environments where RNA-based analyses often fail. IMPORTANCE The novel e- and iDNA separation technique offers insights into the living community at the cell extraction level in the hyperarid Atacama Desert. This approach provides a new framework for analyzing the composition and structure of the potentially active part of the microbial communities as well as their specialization, ecological network and community assembly process. Our findings underscore the significance of utilizing alternative genomic techniques in low-biomass environments where traditional DNA- and RNA-based analyses may not be feasible. The results demonstrate the viability of the proposed study framework and show that specialized microorganisms are important in initial soil formation processes, including microbial-driven mineral weathering, as well as the fixation of carbon and nitrogen.

Acetic acid is a byproduct of lignocellulose pretreatment and a potent inhibitor of yeast-based fermentation processes. A thicker yeast plasma membrane (PM) is expected to retard the passive diffusion of undissociated acetic acid into the cell. Molecular dynamic simulations suggest that membrane thickness can be increased by elongating glycerophospholipids (GPL) fatty acyl chains. Previously, we successfully engineered Saccharomyces cerevisiae to increase GPL fatty acyl chain length but failed to lower acetic acid net uptake. Here, we tested whether altering the relative abundance of diacylglycerol (DAG) might affect PM permeability to acetic acid in cells with longer GPL acyl chains (DAG EN ). To this end, we expressed diacylglycerol kinase α ( DGKα ) in DAG EN . The resulting DAG EN _Dgkα strain exhibited restored DAG levels, grew in medium containing 13 g/L acetic acid, and accumulated less acetic acid. Acetic acid stress and energy burden were accompanied by increased glucose uptake in DAG EN _Dgkα cells. Compared to DAG EN , the relative abundance of several membrane lipids changed in DAG EN _Dgkα in response to acetic acid stress. We propose that the ability to increase the energy supply and alter membrane lipid composition could compensate for the negative effect of high net acetic acid uptake in DAG EN _Dgkα under stressful conditions. IMPORTANCE In the present study, we successfully engineered a yeast strain that could grow under high acetic acid stress by regulating its diacylglycerol metabolism. We compared how the plasma membrane and total cell membranes responded to acetic acid by adjusting their lipid content. By combining physiological and lipidomics analyses in cells cultivated in the absence or presence of acetic acid, we found that the capacity of the membrane to adapt lipid composition together with sufficient energy supply influenced membrane properties in response to stress. We suggest that potentiating the intracellular energy system or enhancing lipid transport to destination membranes should be taken into account when designing membrane engineering strategies. The findings highlight new directions for future yeast cell factory engineering.