Tilling under is a method growers employ in an effort to remediate instances of widespread contamination in fields of crops, but its effectiveness with respect to specific pathogens remains poorly understood. This study had two overarching goals: first, to assess the survival dynamics of Cryptosporidium parvum and Eimeria tenella (used as a surrogate for Cyclospora cayetanensis) oocysts, and Escherichia coli TVS355 in soil following inoculation onto plant tissue and a simulated "tilling under" process; and second, to evaluate the potential of monitoring mRNA levels over time as oocysts die off as an alternative to traditional infectivity-based methods to determine oocyst viability. Basil, cilantro, and soil without plant tissue were inoculated with C. parvum (106 oocysts), E. tenella (106 oocysts), and E. coli TVS355 (108 CFU) and then incorporated into 120 g of soil; samples were then incubated at 12°C or 32°C for up to 12 months. Monthly, samples were removed and assessed for organism survival, E. coli TVS355 through traditional dilution and plating, and oocyst decay through two methods: cell culture followed by qPCR, and mRNA extraction followed by RT-qPCR to measure gene expression. E. coli remained detectable in samples for 10 months, and, through infectivity methods, C. parvum and E. tenella remained infectious for 9 months. However, results from mRNA methods indicated that oocysts remained detectable for at least 12 months, with approximately 1.22 log oocysts/sample detected at that point for both C. parvum and E. tenella. Temperature impacted E. coli TVS355 and E. tenella recovery (as determined by infectivity methods) only after 7 months, when slightly more of each organism was recovered from samples incubated at 12°C. Meanwhile, mRNA methods detected significantly (P < 0.05) fewer oocysts of both C. parvum and E. tenella in samples incubated at 32°C as compared to those incubated at 12°C throughout the experiment.IMPORTANCEContaminated crops may be tilled under and buried in the soil, which may lead to the decay of contaminating microbes. This study looked at three microbes that can contaminate produce: Escherichia coli (a bacterium), Cryptosporidium parvum (a zoonotic protozoan pathogenic parasite), and Eimeria tenella (an avian protozoan parasite used as a stand-in for the human parasite Cyclospora cayetanensis). Inoculated basil and cilantro were mixed in soil and stored at 12°C and 32°C for 1 year. Both protozoan parasites remained infectious for 9 months, and E. coli remained viable for 10 months. Protozoa genetic material and mRNA, as a sign of metabolic activity, were also assessed over time. The type of plant tilled under did not make a meaningful difference. Tilling contaminated crops into soil does not quickly eliminate pathogens. Some pathogens can survive for many months, meaning risks remain in fields for longer than expected.

Well-characterized induction systems that offer tightly regulated, graded, and homogeneous control of gene expression are essential tools for basic and applied microbiology. Tetracycline-based induction systems are widely used non-toxic expression systems in bacteria; however, the generally recognized as safe (GRAS) and industrially relevant model organism Bacillus subtilis currently lacks a tightly regulated, sensitive system that is robust to media and growth conditions. Here, we adapted features of existing tetracycline induction systems to the specific requirements of B. subtilis by engineering a codon-harmonized tetR repressor, enhanced promoter and operator architecture, and a modular shuttle vector enabling stable, single-copy chromosomal integration. The resulting system achieves ~1,000-fold dynamic range with minimal leakiness, using subinhibitory concentrations of anhydrotetracycline, chlortetracycline, and doxycycline as inducers in rich and minimal media as well as in colonies. We further quantify the titration parameters and non-inhibitory ranges for each inducer and growth condition, providing a well-characterized and versatile tool for inducible gene expression in B. subtilis. We also demonstrate that this system can be used to titrate sgRNA expression and associated CRISPRi activity, demonstrating compatibility with other control systems.IMPORTANCEBacillus subtilis is an important bacterial model organism with applications in biotechnology and industrial microbiology. However, tools for tightly controlled gene regulation that are robust to growth conditions remain limited. We developed a tetracycline-inducible system tailored to B. subtilis, providing strong, tunable expression with minimal leaky expression across diverse growth conditions. Due to its enhanced sensitivity, tight regulation, and homogeneous induction of the bacterial population, it can enable precision titration of gene expression for a variety of downstream applications such as physiology studies, pathway optimization, biotechnology, and colony-based screening.

The control of infectious diseases depends on effective diagnostics and interventions. In Africa, resource limitations hinder clinical surveillance. Environmental surveillance (ES), particularly wastewater surveillance, offers a cost-effective alternative. While globally expanding, its application in Africa remains limited. This review aimed to describe published studies in Africa that have utilized ES for the detection of infectious pathogens of public health importance in Africa. The study employed a rapid review approach to synthesize evidence on ES of infectious pathogens in Africa, following guidance from the Cochrane Rapid Reviews Methods. Articles from major databases, including Scopus, PubMed, Science Direct, and Cochrane, were screened using Catchii.org. Duplicates were removed, and data were extracted into Excel, and study quality was appraised using the AXIS tool and a modified Newcastle-Ottawa Scale. The search strategy identified 2,189 articles, of which 90 were found to be eligible. We identified 47 microbial species that have been reported across studies. These consisted of 46.8% bacteria (n = 22), 36.2% viruses (n = 17), 4.3% fungi (n = 2), and 12.7% parasites (n = 6). Among viruses identified, SARS-CoV-2 was the most common, followed by rotaviruses and polioviruses. Vibrio cholerae was mostly reported among bacterial pathogens. The most common sampling method was grab sampling (n = 85, 94.4%), while two-phase separation (n = 22, 37.3%) and filtration (n = 11, 39.3%) were the most frequently used concentration methods for viral and bacterial detection, respectively. ES of infectious pathogens in Africa remains limited. There is a need to expand this to enhance pathogen monitoring, transmission insights, and preparedness for emerging variants.

Microbial homeostasis is maintained by the antagonistic capacity of commensal bacteria against cariogenic pathogens. In the oral cavity, commensal Streptococcus, dominant colonizers of the tooth surface, can produce hydrogen peroxide (H2O2), modulating virulent cross-kingdom biofilm formation. To investigate their ecological role, clinical isolates from dental plaque were compared with reference strains, including Streptococcus oralis ATCC 35037 and S. oralis subsp. tigurinus J22 to determine their H2O2-producing capabilities. The antagonistic potential of S. oralis against Streptococcus mutans and Candida albicans was evaluated using microbial and biochemical assessments. In a saliva-coated hydroxyapatite disc model, S. oralis strains were co-cultured with S. mutans and C. albicans. A high H2O2-producing S. oralis J22 inhibited EPS formation in S. mutans and yeast-to-hypha transition in C. albicans, thereby reducing EPS-mediated bacterial-fungal cell colocalization. Time-lapse confocal imaging revealed that S. oralis J22 dominated the biofilm through H2O2-mediated antagonistic interactions. In contrast, the inhibitory effect of S. oralis strains lacking the spxB gene on cross-kingdom biofilms was significantly reduced. These data provide ecological insights into how physicochemical properties of early colonizing commensals shape the structure and virulence of cross-kingdom oral biofilm through antimicrobial-mediated antagonistic activity.IMPORTANCEThe co-existence of S. mutans and C. albicans accelerates the development of severe early childhood caries, particularly under frequent sucrose exposure. This study demonstrates that early colonizing and antimicrobial-producing oral commensal bacteria can disrupt these pathogenic interactions by modulating their physicochemical associations. These findings highlight the potential of enhancing commensal bacteria as part of novel caries prevention strategies. Further characterization of the functional oral microbiota, especially clinically relevant oral commensals, could advance the development of diagnostic biomarkers and microbiome-targeted therapeutics to prevent painful and costly oral diseases.

Phthalate esters (PAEs) are widely present in the environment, with carcinogenic, teratogenic, and mutagenic toxicity to the human body. The efficient microbial degradation of PAEs is urgent for eco-friendly bioremediation. In addition to PAE esterases, membrane proteins for PAE transport are also important for the microbial degradation of PAEs. However, few experimental reports on the membrane proteins involved in PAE transport, and specifically no studies regarding their underlying transport mechanisms, have been published. Therefore, investigation of the PAE transport mechanisms is crucial for understanding how PAEs enter and exit cells, and it contributes to identifying the rate-limiting steps in PAE degradation. It is conducive to revealing the role of membrane proteins in PAEs degradation, for improving PAE degradation efficiency via membrane protein engineering, or endowing other chassis cells with PAEs degradation capability via constructing membrane protein-esterase co-expressing strains.

Hydroxytyrosol (HT), the primary functional component of olive oil, presents significant challenges for microbial biosynthesis due to its inherent chemical properties and fermentation requirements. In this study, we describe the modular engineering of HT production in Escherichia coli (E. coli). We initially employed a highly efficient 4-hydroxyphenylacetic acid 3-monooxygenase (encoded by the HpaBC gene) from E. coli, phenylpyruvate decarboxylase (encoded by the ARO10 gene), and alcohol dehydrogenase (encoded by the ADH6 gene) from Saccharomyces cerevisiae (S. cerevisiae) to establish the de novo biosynthetic pathway for HT in E. coli. To enhance carbon flux toward HT production, we attenuated the competing metabolic pathway while reinforcing the synthetic route and optimized the gene copy numbers of key enzymes involved. Further strain engineering involved strengthening the membrane-bound pyridine nucleotide transhydrogenase (encoded by the pntAB gene) and incorporating a heterologous riboflavin biosynthesis pathway to improve the supply of the cofactors NADPH and FADH₂, culminating in the generation of the high-producing strain HT32-3. Then, a two-stage potential of hydrogen (pH) and two-stage dissolved oxygen (DO) control strategy was implemented to mitigate oxidative degradation of HT. In addition, a fed-batch supplementation process was developed to address the dependency of the key enzyme ARO10 on the cofactor vitamin B₁ (VB₁). These combined strategies enabled the achievement of a final HT titer of 9.22 g/L in a 5 L bioreactor. The present study provides a practical strategy for the modification of HT strains and a fermentation strategy, which lays the foundation for the development of engineered strains of high-value derivatives of tyrosine.IMPORTANCEIn this study, a strain with high hydroxytyrosol (HT) production capacity was constructed by means of metabolic engineering modification. A two-stage pH and two-stage DO fermentation strategy was developed based on its physicochemical properties. Combined with the VB1 replenishment strategy, the fermentation process of this strain was optimized, and the retention of HT was significantly improved, laying the foundation for the large-scale production of HT. This study explored the metabolic synthesis pathway and efficient fermentation strategy of HT, providing an innovative fermentation strategy and a practical strategy for the fermentation and production of HT and its related products.

The persistent and deadly cholera remains a significant burden on global health. Current countermeasures are inadequate against cholera. Toxin-coregulated pilin (TCP) has long been a target for understanding the biogenesis of Vibrio cholerae infection and for developing subunit vaccines against cholera. In this study, we examined TcpA B-cell epitopes of the O1 classical and El Tor biotypes, as well as the O139 serogroup, for functionality, potential biogenesis, and vaccine application. By empirically mapping TcpA functional epitopes across V. cholerae biotypes and serogroups, we found that antibodies raised against a shared epitope (EP #1, IQSQNMTKAAQ) provided partial protection against V. cholerae bacterial adherence in vitro. Data also showed that classical biotype TcpA epitope #6, ASKNLDLTNITHVE, is more effective in inducing functional antibodies against adherence of the classical biotype, but not against the El Tor biotype. Similarly, El Tor biotype TcpA epitopes #4 (FAAVADLGDFETSV) and #6 (GSANLNLTNITHVE) are effective in inducing functional antibodies against the adherence of El Tor biotype and O139 serogroup, but not against the classical biotype. This decodes early observations of a lack of cross-protection from TcpA-derived immunity. Interestingly, combined immunity raised against the classical epitope #6 and the El Tor epitope #6 (or #4) effectively protected against adherence from both biotypes. These results reveal the mechanism underlying the lack of protective immunity conferred by biotype-specific TcpA and indicate a combined epitope immunity for protection across biotypes and serogroups, providing a better understanding of TcpA immunity, perhaps its biogenesis, and guiding the development of cholera vaccines.IMPORTANCECurrent countermeasures are inadequate against cholera, a disease that remains a significant global health burden. Oral cholera vaccines (OCVs) prequalified by the World Health Organization (WHO) are serogroup-specific. In addition to the supply shortage, these whole-cell vaccines provide limited protection to children under 5 years of age in endemic countries, the most vulnerable population to cholera, urgently calling for more effective prevention strategies. By examining functional immunity raised against shared and biotype-specific TcpA epitopes, this study revealed the mechanism underlying why TCP-related immunity from the O1 classical biotype does not extend protection to the O1 El Tor biotype. The current study also showed that a combined biotype-epitope immunity provides cross-protection against O1 El Tor and classical biotypes, as well as against the O139 serogroup, potentially paving the way for the development of a cross-protective vaccine against Vibrio cholerae infection.

Mucinases produced by the gut microbiota play a dual role in regulating the integrity and renewal of the mucus layer, which is essential for maintaining gut homeostasis and human health. In this study, we constructed protein hidden Markov models based on 11 known mucinases and used them to systematically identify mucinase sequences from gut metagenome-assembled genomes derived from 80 colorectal cancer (CRC) patients and 86 healthy (Healthy) subjects. A total of 1,869 mucinases were detected, widely distributed across the studied cohorts, with the majority originating from Bacteroides, Phocaeicola, and Akkermansia species. Further analysis identified 42 mucinases that differed significantly in abundance between the two groups, all of which were enriched in CRC patients. Taxonomic attribution revealed that, in CRC patients, these mucinases were primarily derived from Bacteroides (36.0%), Phocaeicola (30.6%), Akkermansia (8.8%), Alistipes (8.6%), and Escherichia (6.4%), whereas in Healthy subjects, they mainly originated from Bacteroides (26.1%), Akkermansia (22.7%), and Phocaeicola (20.3%), with a notably higher proportion from Akkermansia. Among the 42 mucinases, WL42 and LLN1 exhibited significantly higher abundance levels compared to the others. Phylogenetic and predicted structural analyses suggested that these two mucinases belonged to the M60 and M98 families, respectively. Functional validation through co-incubation experiments demonstrated that both mucinases could cleave the glycosylated MUC1 and MUC2 substrates, but not the corresponding non-glycosylated proteins. These findings confirm the feasibility of discovering novel mucinases directly from gut metagenomic data and provide insights into their potential roles in health and disease.IMPORTANCEOur study established a feasible bioinformatics pipeline for the systematic identification of microbial mucinases within the gut microbiome, providing a methodological foundation for large-scale mining of functionally active mucin-degrading enzymes. We identified 42 mucinases significantly enriched in CRC patients, suggesting their potential involvement in CRC pathogenesis. Among them, two mucinases were experimentally validated for their ability to degrade mucin, offering direct functional evidence of their capacity to disrupt the mucosal barrier. Genus-level metagenomic profiling further identified Bacteroides, Phocaeicola, and Akkermansia as major mucinase-producing genera. Maintaining the secretory balance of these mucinase-producing bacteria might be crucial for ameliorating intestinal barrier dysfunction in CRC patients. The findings of this study offer critical insights into the microbial origins and potential mechanistic contributions of mucinases in colorectal cancer, underscoring their relevance in mucus barrier breakdown and disease progression.

Mycoplasmas are reliant on host cells for the acquisition of nutrients, and their metabolic functions are vital for both survival and virulence. Metabolomic analysis can be used to determine the metabolic functions of genes in mycoplasmas by examining the metabolic changes in mutants with gene disruptions. We combined different metabolomic techniques to study the metabolic changes in the important bovine pathogen Mycoplasma bovis caused by the disruption of the putative deoC gene, which is essential for the survival of this organism in association with host cells. Our results suggest the product of the gene plays a role in the catabolism of deoxyribonucleosides and that this metabolic pathway is critically important in the interactions of M. bovis with host cells.

Rhizobia are soil bacteria that establish symbiotic associations with legumes, converting atmospheric nitrogen into ammonia through biological nitrogen fixation, while the host provides nutrients. Among them, Sinorhizobium meliloti is one of the best-studied species. In this work, we compared the complete genomes of S. meliloti strains, including the laboratory model strain LPU88, with a particular focus on pSymA plasmids. Previous studies proposed that the pSymA plasmid could have been acquired through horizontal gene transfer. Analysis of their conjugation machinery revealed that all pSymA plasmids harbor a type II conjugation system, although in many cases the regulatory circuit required for activation was absent. In LPU88, we identified and characterized multiple conjugation systems, offering new insights into horizontal gene transfer in S. meliloti. Understanding these processes is essential for clarifying rhizobial evolutionary dynamics, improving the stability and efficiency of symbiotic interactions, and promoting their use as bioinoculants in sustainable agriculture.