Improved bacterial recombineering by parallelized protein discovery.

Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found. Here we introduce a high-throughput method for SSAP discovery that we call "serial enrichment for efficient recombineering" (SEER). By performing SEER in Escherichia coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5- to 10-fold in E. coli, while PapRecT enables efficient recombineering in Pseudomonas aeruginosa, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.

RecT Affects Prophage Lifestyle and Host Core Cellular Processes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a notorious pathogen that causes various nosocomial infections. Several prophage genes located on the chromosomes of P. aeruginosa have been reported to contribute to bacterial pathogenesis via host phenotype transformations, such as serotype conversion and antibiotic resistance. However, our understanding of the molecular mechanism behind host phenotype shifts induced by prophage genes remains largely unknown. Here, we report a systematic study around a hypothetical recombinase, Pg54 (RecT), located on a 48-kb putative prophage (designated PP9W) of a clinical P. aeruginosa strain P9W. Using a ΔrecT mutant (designated P9D), we found that RecT promoted prophage PP9W excision and gene transcription via the inhibition of the gene expression level of pg40, which encodes a CI-like repressor protein. Further transcriptomic profiling and various phenotypic tests showed that RecT modulated like a suppressor to some transcription factors and vital genes of diverse cellular processes, providing multiple advantages for the host, including cell growth, biofilm formation, and virulence. The versatile functions of RecT hint at a strong impact of phage proteins on host P. aeruginosa phenotypic flexibility. IMPORTANCE Multidrug-resistant and metabolically versatile P. aeruginosa are difficult to eradicate by anti-infective therapy and frequently lead to significant morbidity and mortality. This study characterizes a putative recombinase (RecT) encoded by a prophage of a clinical P. aeruginosa strain isolated from severely burned patients, altering prophage lifestyle and host core cellular processes. It implies the potential role of RecT in the coevolution arm race between bacteria and phage. The excised free phages from the chromosome of host bacteria can be used as weapons against other sensitive competitors in diverse environments, which may increase the lysogeny frequency of different P. aeruginosa subgroups. Subsequent analyses revealed that RecT both positively and negatively affects different phenotypic traits of the host. These findings concerning RecT functions of host phenotypic flexibility improve our understanding of the association between phage recombinases and clinical P. aeruginosa, providing new insight into mitigating the pathogen infection.

Improved acid-stress tolerance of Lactococcus lactis NZ9000 and Escherichia coli BL21 by overexpression of the anti-acid component recT.

Acid accumulation caused by carbon metabolism severely affects the fermentation performance of microbial cells. Here, different sources of the recT gene involved in homologous recombination were functionally overexpressed in Lactococcus lactis NZ9000 and Escherichia coli BL21, and their acid-stress tolerances were investigated. Our results showed that L. lactis NZ9000 (ERecT and LRecT) strains showed 1.4- and 10.4-fold higher survival rates against lactic acid (pH 4.0), respectively, and that E. coli BL21 (ERecT) showed 16.7- and 9.4-fold higher survival rates than the control strain against lactic acid (pH 3.8) for 40 and 60 min, respectively. Additionally, we found that recT overexpression in L. lactis NZ9000 improved their growth under acid-stress conditions, as well as increased salt- and ethanol-stress tolerance and intracellular ATP concentrations in L. lactis NZ9000. These findings demonstrated the efficacy of recT overexpression for enhancing acid-stress tolerance and provided a promising strategy for insertion of anti-acid components in different hosts.

Construction of a bacteriophage-derived recombinase system in Bacillus licheniformis for gene deletion.

Bacillus licheniformis and its related strains have found extensive applications in diverse industries, agriculture, and medicine. However, the current breeding methods for this strain primarily rely on natural screening and traditional mutagenesis. The limited availability of efficient genetic engineering tools, particularly recombination techniques, has hindered further advancements in its applications. In this study, we conducted a comprehensive investigation to identify and characterize a recombinase, RecT, derived from a Bacillus phage. Remarkably, the recombinase exhibited a 105-fold enhancement in the recombination efficiency of the strain. To facilitate genome editing, we developed a system based on the conditional expression of RecT using a rhamnose-inducible promoter (Prha). The efficacy of this system was evaluated by deleting the amyL gene, which encodes an α-amylase. Our findings revealed that the induction time and concentration of rhamnose, along with the generation time of the strain, significantly influenced the editing efficiency. Optimal conditions for genome editing were determined as follows: the wild-type strain was initially transformed with the genome editing plasmid, followed by cultivation and induction with 1.5% rhamnose for 8 h. Subsequently, the strain was further cultured for an additional 24 h, equivalent to approximately three generations. Consequently, the recombination efficiency reached an impressive 16.67%. This study represents a significant advancement in enhancing the recombination efficiency of B. licheniformis through the utilization of a RecT-based recombination system. Moreover, it provides a highly effective genome editing tool for genetic engineering applications in this strain.

[Resident and supervisor perception of learning climate in public health resident training in the Netherlands]. / Leerklimaat perceptie van aios en supervisors binnen de vernieuwde medische vervolgopleiding arts Maatschappij + Gezondheid.

Introduction: The purpose of this study was to evaluate the perceived quality of the learning climate by public health residents in the Netherlands and compare residents' and supervisors' perceptions. Methods: Residents of five public health subfields, who started their residency programs in 2019 and onwards, as well as supervisors involved in the residency program, were invited to complete a web-based survey based on an adapted version of the D­RECT questionnaire. Answers of residents and supervisors of the same training site and public health subfield were matched to compare perceived quality of the learning climate. Results: One hundred fourteen residents responded (response rate 50.9%). Residents' overall assessment of the learning climate showed a mean score of 4.19 on a 5-point-scale. Thirty-eighth supervisor-resident matches were formed. There were no notable differences in the perception of residents and supervisors. Conclusion: Residents' overall assessment of the learning climate was positive. Supervisors and residents' perception of learning climate is equal. Our adapted version of D­RECT seems to be suitable to evaluate the learning climate for public health residency programs in the Netherlands. Further research is necessary to validate our questionnaire and to confirm our findings.

CRISPR-Cas12a System With Synergistic Phage Recombination Proteins for Multiplex Precision Editing in Human Cells.

The development of CRISPR-based gene-editing technologies has brought an unprecedented revolution in the field of genome engineering. Cas12a, a member of the Class 2 Type V CRISPR-associated endonuclease family distinct from Cas9, has been repurposed and developed into versatile gene-editing tools with distinct PAM recognition sites and multiplexed gene targeting capability. However, with current CRISPR/Cas12a technologies, it remains a challenge to perform efficient and precise genome editing of long sequences in mammalian cells. To address this limitation, we utilized phage recombination enzymes and developed an efficient CRISPR/Cas12a tool for multiplexed precision editing in mammalian cells. Through protein engineering, we were able to recruit phage recombination proteins to Cas12a to enhance its homology-directed repair efficiencies. Our phage-recombination-assisted Cas12a system achieved up to 3-fold improvements for kilobase-scale knock-ins in human cells without compromising the specificity of the enzyme. The performance of this system compares favorably against Cas9 references, the commonly used enzyme for gene-editing tasks, with improved specificity. Additionally, we demonstrated multi-target editing with similar improved activities thanks to the RNA-processing activity of the Cas12a system. This compact, multi-target editing tool has the potential to assist in understanding multi-gene interactions. In particular, it paves the way for a gene therapy method for human diseases that complements existing tools and is suitable for polygenic disorders and diseases requiring long-sequence corrections.

The Dutch residency educational climate test: construct and concurrent validation in Spanish language.

OBJECTIVES: To translate the 35-item version of the Dutch Residency Educational Climate Test (D-RECT), and assess its reliability, construct validity and concurrent validity in the Spanish language. METHODS: For this validation study, the D-RECT was translated using international recommendations. A total of 220 paper-based resident evaluations covering two Colombian universities were cross-sectionally collected in 2015. A Confirmatory Factor Analysis (CFA) was used to assess the internal validity of the instrument using the Comparative fit index (CFI), Tucker-Lewis index (TLI), Standardized root mean square residual (SRMSR), and Root mean square error of approximation (RMSA). Cronbach's α was used to assess reliability. The concurrent validity was investigated through Pearson correlations with the Spanish version of the Postgraduate Hospital Educational Environment Measure (PHEEM). RESULTS: The original 9-factor structure showed an appropriate fit for the Spanish version of the instrument (CFI = 0.84, TLI = 0.82, SRMSR = 0.06, and RMSA = 0.06). The reliability coefficients were satisfactory (>0.70). The mean total scores of the D-RECT and the PHEEM showed a significant correlation (r = 0.7, p<0.01). CONCLUSIONS: This study confirms the validity and reliability of the Spanish version of the Dutch Residency Educational Climate Test, indicating that the instrument is suitable for the evaluation of departments' learning climate in the Spanish context. Future research is needed to confirm these findings in other Spanish speaking countries.

Learning climate and quality of Italian training courses in gynecology and obstetrics.

OBJECTIVES: To evaluate the learning climate (LC) and quality of training in postgraduate training courses in gynecology and obstetrics in Italy, as essential element to improve the training quality of future medical specialists. STUDY DESIGN: Web-based anonymous survey sent to all Italian trainees in gynecology and obstetrics to assess LC and quality of postgraduate training courses. This included sociodemographic information, details regarding training positions, and a 50-item validated Dutch Residency Educational Climate Test (D-RECT) questionnaire with 11 subscales (1-5 Likert scale). At the same time, the 24-items Fifth Year Training Questionnaire (FYT-Q) was submitted to all trainees at the fifth year of training to assess quality of life (burnout and depression), quality of training and final achieved competency level. Descriptive statistics were used to describe the main characteristics of the study population and for the D-RECT and the FYT-Q results. RESULTS: One hundred seventy-eight trainees' responses were included from 13 departments, yielding a department response rate of 33%. The mean composite score of the D-RECT was 3.185 (SD 0.305). The subscales "Formal education" and "Role of specialty tutor" scored a mean of 2.751 (SD 0.123) and 2.757 (SD 0.130), respectively. Sixty-four FYT-Q evaluations were completed. The 33% of trainees reported more than 56 weekly working hours. At least one burnout episode during the training was reported by 61% of the trainees, and the 45% of them reported one or more episode of depression. More than 50% of trainees reported adequate autonomy for gynecologic ultrasound, obstetrics first level ultrasound, hysteroscopy, and cesarean section. In FYT-Q adequacy of training, teaching, surgical teaching, and tutoring values resulted equal to or less than 3 in a 1-5 Likert scale. CONCLUSIONS: D-RECT and FYT-Q questionnaires show a training that requires improvement, although the results do not seem to be completely consistent. D-RECT emphasizes the need for a better formal teaching and specialty tutors to ensure training with better LC. Interventions are needed to improve LC and quality of training in postgraduate training courses in gynecology and obstetrics in Italy.

The Questionnaire D-RECT German: Adaptation and testtheoretical properties of an instrument for evaluation of the learning climate in medical specialist training.

AIM: Boor et al developed and validated the questionnaire D-RECT (Dutch Residency Educational Climate Test ) to measure the clinical learning environment within the medical specialist training. In this study, a German version of this questionnaire (D-RECT German) is analyzed regarding testtheoretical properties. PROBLEM: Are the results of Boor et al replicable as a proof for validity of the questionnaire D-RECT? MATERIAL & METHODS: The study was performed as online survey using the questionnaire D-RECT German (50 items in 11 subscales). To determine item characteristics and internal consistency (Cronbach's α), item- and reliability analyses were performed. Furthermore, a confirmatory factor analysis was performed using a model for maximum-likelihood estimation to evaluate validity. RESULTS: This replication study on the psychometric properties of the D-RECT with 255 residents at 17 German hospitals revealed heterogeneous discriminatory power for all items and an internal consistency of Cronbach's α between 0.57 and 0.85. Within the confirmatory factor analysis, 6 items showed standardized regression coeffizients <0.5, two of them in the subscale "Attendings role". Furthermore, strong interdependencies (>0.7) were found between the subscales "Supervision", "Coaching" and "Attendings role". CONCLUSION: The present replication study with the D-RECT German showed structural differences with respect to factorial validity underpinning the need of further validation studies.

A functional recT gene for recombineering of Clostridium.

Recombineering is an efficient genetic manipulation method employing the mechanism of phagenic RecT-mediated homologous recombination. To develop a recombineering method for Clostridium, a putative recT gene (CPF0939) from Clostridium perfringens genome was functionally verified in a clostridial host Clostridium acetobutylicum. We show that a short synthetic oligonucleotide can be introduced into the target site for specific point mutation. This functional recT gene would therefore contribute to development of recombineering tools for Clostridium.