Engineering artificial cross-species promoters with different transcriptional strengths.

As a fundamental tool in synthetic biology, promoters are pivotal in regulating gene expression, enabling precise genetic control and spurring innovation across diverse biotechnological applications. However, most advances in engineered genetic systems rely on host-specific regulation of the genetic portion. With the burgeoning diversity of synthetic biology chassis cells, there emerges a pressing necessity to broaden the universal promoter toolkit spectrum, ensuring adaptability across various microbial chassis cells for enhanced applicability and customization in the evolving landscape of synthetic biology. In this study, we analyzed and validated the primary structures of natural endogenous promoters from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Pichia pastoris, and through strategic integration and rational modification of promoter motifs, we developed a series of cross-species promoters (Psh) with transcriptional activity in five strains (prokaryotic and eukaryotic). This series of cross species promoters can significantly expand the synthetic biology promoter toolkit while providing a foundation and inspiration for standardized development of universal components The combinatorial use of key elements from prokaryotic and eukaryotic promoters presented in this study represents a novel strategy that may offer new insights and methods for future advancements in promoter engineering.

Phosphonic Acids as Corrosion Inhibitors and Adhesion Promoters for Organic Coatings and Bronze.

Currently used organic coatings for the protection of bronze sculptures have a relatively short lifespan as a consequence of strict requirements of conservation ethics, which limit the selection of coatings. For that reason, enhancement of the corrosion protection level and durability of appropriate coatings is needed. The aim of this work was to examine if corrosion protection of bronze by selected acrylic and polyurethane coatings could be improved by using two phosphonic acids, 16-phosphonohexadecanoic acid (COOH-PA) and 12-aminododecylphosphonic acid (NH2-PA). Electrochemical measurements (linear polarization and electrochemical impedance spectroscopy, EIS) were performed to gain an insight into the influence of these phosphonic acids on the performance of the coatings during a two-week exposure to artificial acid rain and a three-month outdoor exposure. Besides the influence on the corrosion protection level, the influence on the coating adhesion was examined as well. A pull-off test clearly confirmed that the studied phosphonic acids act as adhesion promoters of both polyurethane and acrylic coatings, while electrochemical studies revealed improvements in corrosion protection levels, especially in the case of the acrylic coating Paraloid B72.

A new approach: preventive protocols with yeast products and essential oils can reduce the in-feed use of antibiotics in growing-finishing pigs.

The objective of this study was to evaluate the effects of yeast products (YP) and essential oils (EO) in total or partial replacement to in-feed antibiotic protocols (growth promoter and prophylactic), both in recommended doses and in overdose of prophylactic antibiotics (PA), on growth performance, and diarrhea incidence in the growing-finishing pigs; and fecal microbiota in market hogs. Four hundred pigs (20.36 ±â€…2.64 kg) were assigned to five treatments in a randomized block design: diets with prophylactic and growth promoter antibiotics (ANT); ANT with 30% more PA (ANT+30); diets with less PA and YP (ANT+Y); diets with less PA, YP and EO (ANT+Y+EO); and antibiotics-free diets with YP and EO (Y+EO). The content of the active components of the YP was 60% purified ß-1,3/1,6-glucans extracted from Saccharomyces cerevisiae yeast (Macrogard), 20% functional water-soluble MOS (HyperGen), and 18% MOS, extracted from Saccharomyces cerevisiae yeast (ActiveMOS). From 0 to 14 d, pigs of the ANT+30, ANT+Y, and ANT+Y+EO treatments showed a greater body weight (BW) and average daily gain (ADG) compared to pigs from the Y+EO group. From 14 to 35 d, pigs of ANT+30 and ANT+Y+EO treatments were heavier than Y+EO group. At 105 d, ANT pigs had a higher BW than the Y+EO group. For the entire period, ADG of ANT pigs was greater, and feed conversion ratio better than Y+EO pigs. From 0 to 35 d, pigs of the Y+EO treatment showed a higher diarrhea incidence compared to pigs of the other groups. From 49 to 70 d, ANT+Y and ANT+Y+EO treatments showed a lower diarrhea incidence than Y+EO group, which remained the case during the overall period. At 105 d, the alpha diversity of fecal microbiota by Shannon Entropy was lower in ANT, ANT+30, and Y+EO groups than observed for ANT+Y+EO group. The abundance of Firmicutes phylum and Firmicutes/Bacteroidetes ratio was higher in ANT than in ANT+Y+EO pigs. Proteobacteria phylum abundance in ANT+Y+EO was higher than ANT, ANT+Y, and Y+EO. Peptostreptococcaceae family abundance was higher in ANT, ANT+30, and ANT+Y groups than in ANT+Y+EO and Y+EO groups. ANT+Y+EO and Y+EO groups show a lower abundance of SMB53 genus than ANT and ANT+30 groups. In conclusion, the use of YP and EO, in partial replacement to the in-feed antibiotic protocols, does not reduce the growth performance, can replace antibiotic growth promotors, and reduce the in-feed use of PA in growing-finishing pigs. The use of YP and EO, together with PA, increases the microbial diversity, despite having important genera for weight gain in less abundance. Overdose of PA does not improve growth performance and reduces microbial diversity, which does not characterize it as an efficient preventive protocol.

A novel deep learning identifier for promoters and their strength using heterogeneous features.

Promoters, which are short (50-1500 base-pair) in DNA regions, have emerged to play a critical role in the regulation of gene transcription. Numerous dangerous diseases, likewise cancer, cardiovascular, and inflammatory bowel diseases, are caused by genetic variations in promoters. Consequently, the correct identification and characterization of promoters are significant for the discovery of drugs. However, experimental approaches to recognizing promoters and their strengths are challenging in terms of cost, time, and resources. Therefore, computational techniques are highly desirable for the correct characterization of promoters from unannotated genomic data. Here, we designed a powerful bi-layer deep-learning based predictor named "PROCABLES", which discriminates DNA samples as promoters in the first-phase and strong or weak promoters in the second-phase respectively. The proposed method utilizes five distinct features, such as word2vec, k-spaced nucleotide pairs, trinucleotide propensity-based features, trinucleotide composition, and electron-ion interaction pseudopotentials, to extract the hidden patterns from the DNA sequence. Afterwards, a stacked framework is formed by integrating a convolutional neural network (CNN) with bidirectional long-short-term memory (LSTM) using multi-view attributes to train the proposed model. The PROCABLES model achieved an accuracy of 0.971 and 0.920 and the MCC 0.940 and 0.840 for the first and second-layer using the ten-fold cross-validation test, respectively. The predicted results anticipate that the proposed PROCABLES protocol outperformed the advanced computational predictors targeting promoters and their types. In summary, this research will provide useful hints for the recognition of large-scale promoters in particular and other DNA problems in general.

Oncogenic and telomeric G-quadruplexes: Targets for porphyrin-triphenylphosphonium conjugates.

DNA chains with sequential guanine (G) repeats can lead to the formation of G-quadruplexes (G4), which are found in functional DNA and RNA regions like telomeres and oncogene promoters. The development of molecules with adequate structural features to selectively stabilize G4 structures can counteract cell immortality, highly described for cancer cells, and also downregulate transcription events underlying cell apoptosis and/or senescence processes. We describe here, the efficiency of four highly charged porphyrins-phosphonium conjugates to act as G4 stabilizing agents. The spectrophotometric results allowed to select the conjugates P2-PPh3 and P3-PPh3 as the most promising ones to stabilize selectively G4 structures. Molecular dynamics simulation experiments were performed and support the preferential binding of P2-PPh3 namely to MYC and of P3-PPh3 to KRAS. The ability of both ligands to block the activity of Taq polymerase was confirmed and also their higher cytotoxicity against the two melanoma cell lines A375 and SK-MEL-28 than to immortalized skin keratinocytes. Both ligands present efficient cellular uptake, nuclear co-localization and high ability to generate 1O2 namely when interacting with G4 structure. The obtained data points the synthesized porphyrins as promising ligands to be used in a dual approach that can combine G4 stabilization and Photodynamic therapy (PDT).

The influential responsibility of sirtuins in senescence and associated diseases: A review.

Aging is a process of time-associated depletion in the physiological functions, essential for the survival and reproducibility of living beings. Some age-related disorders can be successfully controlled with some biomedical techniques or pharmaceutical approaches. There are some precise remedies that demonstrate conspicuous promise in the preclinical and clinical setup of extending lifespan or enhancing health by altering natural senescence. The sirtuin family of proteins is one of the most favorable targets for antiaging strategies. Sirtuins were initially identified as transcription repressors in yeast, but today they are known to exist in bacteria and eukaryotes, as well as humans. The SIRT (1-7) family of proteins in humans is made up of seven members, each of which has either mono-ADP ribosyl transferase or deacetylase activity. Researchers suggest that sirtuins are essential for cell metabolism and play a major role in how cells react to various stimuli, such as oxidative or genotoxic stress. A healthy lifestyle, which includes exercise and a balanced diet, has been demonstrated to impact health span by adjusting the levels of sirtuins, suggesting the involvement of sirtuins in extending human longevity. The hunt for sirtuin activators is among the most extensive and comprehensive research subjects in the present scenario. Some optimism has been generated to investigate antiaging therapies by natural compounds, such as curcumin and others. This review article highlights the role of sirtuins in native senescence and their primordial roles in the progression of several life-threatening diseases. Further, it also provides recent information on the sirtuin activators and inhibitors and their therapeutic benefits.

Exploring the Use of Alternative Promoters for Enhanced Transgene and sgRNA Expression in Atlantic Salmon Cells.

This study facilitates design of expression vectors and lentivirus tools for gene editing of Atlantic salmon. We have characterized widely used heterologous promoters and novel endogenous promoters in Atlantic salmon cells. We used qPCR to evaluate the activity of several U6 promoters for sgRNA expression, including human U6 (hU6), tilapia U6 (tU6), mouse U6 (mU6), zebrafish U6 (zU6), Atlantic salmon U6 (sU6), medaka U6 (medU6), and fugu U6 (fU6) promoters. We also evaluated several polymerase type II (pol II) promoters by luciferase assay. Our results showed that hU6 and tU6 promoters were the most active among all the tested U6 promoters, and heterologous promoters (CMV, hEF1α core) had higher activity compared to endogenous Atlantic salmon promoters sHSP8, sNUC3L, sEF1α. Among endogenous pol II promoters, sEF1α and sHSP8 displayed higher activity than sNUC3L, sHSP703, sHSP7C, sXRCC1L, and sETF. We observed that extending the promoter sequence to include the region up to the start codon (ATG) resulted in a significant increase in expression efficiency for sNUC3L and sEF1α. We also show that mutating the PRDM1 motif will significantly decrease the activity of the sEF1α promoter. The presence of the PRDM1 motif in sHSP8 promoter was also associated with relatively high expression compared to the promoters that naturally lacked this motif, such as sNUC3L. We speculate that this short sequence might be included in other promoters to further enhance the promoter activity, but further experiments are needed to confirm this. Our findings provide valuable insights into the activity of different promoters in Atlantic salmon cells and can be used to facilitate further transgenic studies and improve the efficiency of transgene expression in Atlantic salmon.

Synonymous codon substitutions modulate transcription and translation of a divergent upstream gene by modulating antisense RNA production.

Synonymous codons were originally viewed as interchangeable, with no phenotypic consequences. However, substantial evidence has now demonstrated that synonymous substitutions can perturb a variety of gene expression and protein homeostasis mechanisms, including translational efficiency, translational fidelity, and cotranslational folding of the encoded protein. To date, most studies of synonymous codon-derived perturbations have focused on effects within a single gene. Here, we show that synonymous codon substitutions made far within the coding sequence of Escherichia coli plasmid-encoded chloramphenicol acetyltransferase (cat) can significantly increase expression of the divergent upstream tetracycline resistance gene, tetR. In four out of nine synonymously recoded cat sequences tested, expression of the upstream tetR gene was significantly elevated due to transcription of a long antisense RNA (asRNA) originating from a transcription start site within cat. Surprisingly, transcription of this asRNA readily bypassed the native tet transcriptional repression mechanism. Even more surprisingly, accumulation of the TetR protein correlated with the level of asRNA, rather than total tetR RNA. These effects of synonymous codon substitutions on transcription and translation of a neighboring gene suggest that synonymous codon usage in bacteria may be under selection to both preserve the amino acid sequence of the encoded gene and avoid DNA sequence elements that can significantly perturb expression of neighboring genes. Avoiding such sequences may be especially important in plasmids and prokaryotic genomes, where genes and regulatory elements are often densely packed. Similar considerations may apply to the design of genetic circuits for synthetic biology applications.

Unlocking the potential of cyanobacteria: a high-throughput strategy for enhancing biocatalytic performance through genetic optimization.

Cyanobacteria show promise as hosts for whole-cell biocatalysis. Their photoautotrophic metabolism can be leveraged for a sustainable production process. Despite advancements, performance still lags behind heterotrophic hosts. A key challenge is the limited ability to overexpress recombinant enzymes, which also hinders their biocatalytic efficiency. To address this, we generated large-scale expression libraries and developed a high-throughput method combining fluorescence-activated cell sorting (FACS) and deep sequencing in Synechocystis sp. PCC 6803 (Syn. 6803) to screen and optimize its genetic background. We apply this approach to enhance expression and biocatalyst performance for three enzymes: the ketoreductase LfSDR1M50, enoate reductase YqjM, and Baeyer-Villiger monooxygenase (BVMO) CHMOmut. Diverse genetic combinations yielded significant improvements: optimizing LfSDR1M50 expression showed a 17-fold increase to 39.2 U gcell dry weight (CDW)-1. In vivo activity of Syn. YqjM was improved 16-fold to 58.7 U gCDW-1 and, for Syn. CHMOmut, a 1.5-fold increase to 7.3 U gCDW-1 was achieved by tailored genetic design. Thus, this strategy offers a pathway to optimize cyanobacteria as expression hosts, paving the way for broader applications in other cyanobacteria strains and larger libraries.

Sirtuin insights: bridging the gap between cellular processes and therapeutic applications.

The greatest challenges that organisms face today are effective responses or detection of life-threatening environmental changes due to an obvious semblance of stress and metabolic fluctuations. These are associated with different pathological conditions among which cancer is most important. Sirtuins (SIRTs; NAD+-dependent enzymes) are versatile enzymes with diverse substrate preferences, cellular locations, crucial for cellular processes and pathological conditions. This article describes in detail the distinct roles of SIRT isoforms, unveiling their potential as either cancer promoters or suppressors and also explores how both natural and synthetic compounds influence the SIRT function, indicating promise for therapeutic applications. We also discussed the inhibitors/activators tailored to specific SIRTs, holding potential for diseases lacking effective treatments. It may uncover the lesser-studied SIRT isoforms (e.g., SIRT6, SIRT7) and their unique functions. This article also offers a comprehensive overview of SIRTs, linking them to a spectrum of diseases and highlighting their potential for targeted therapies, combination approaches, disease management, and personalized medicine. We aim to contribute to a transformative era in healthcare and innovative treatments by unraveling the intricate functions of SIRTs.

Benzofuran and Benzo[b]thiophene-2-carboxamide Derivatives as Modulators of Amyloid Beta (Aß42) Aggregation.

A group of N-phenylbenzofuran-2-carboxamide and N-phenylbenzo[b]thiophene-2-carboxamide derivatives were designed and synthesized as a novel class of Aß42 aggregation modulators. In the thioflavin-T based fluorescence aggregation kinetics study, compounds 4a, 4b, 5a and 5b possessing a methoxyphenol pharmacophore were able to demonstrate concentration dependent inhibition of Aß42 aggregation with maximum inhibition of 54% observed for compound 4b. In contrast, incorporation of a 4-methoxyphenyl ring in compounds 4d and 5d led to a significant increase in Aß42 fibrillogenesis demonstrating their ability to accelerate Aß42 aggregation. Compound 4d exhibited 2.7-fold increase in Aß42 fibrillogenesis when tested at the maximum concentration of 25 µM. These results were further confirmed by electron microscopy studies which demonstrates the ability of compounds 4a, 4b, 4d, 5a, 5b and 5d to modulate Aß42 fibrillogenesis. Compounds 5a and 5b provided significant neuroprotection to mouse hippocampal neuronal HT22 cells against Aß42-induced cytotoxicity. Molecular docking studies suggest that the orientation of the bicyclic aromatic rings (either benzofuran or benzo[b]thiophene) plays a major role in moderating their ability to either inhibit or accelerate Aß42 aggregation. Our findings support the application of these novel derivatives as pharmacological tools to study the mechanisms of Aß42 aggregation.

Rational Design and Screening of Synthetic Promoters in Chlamydomonas reinhardtii.

Synthetic promoters are powerful tools to boost the biotechnological potential of microalgae as eco-sustainable industrial hosts. The increasing availability of transcriptome data on microalgae in a variety of environmental conditions allows to identify cis-regulatory elements (CREs) that are responsible for the transcriptional output. Furthermore, advanced cloning technologies, such as golden gate-based MoClo toolkits, enable the creation of modular constructs for testing multiple promoters and a range of reporter systems in a convenient manner. In this chapter, we will describe how to introduce in silico-identified CREs into promoter sequences, and how to clone the modified promoters into MoClo compatible vectors. We will then describe how these promoters can be evaluated and screened for transgene expression in an established microalgal model for genetic perturbation, i.e., Chlamydomonas reinhardtii.

Chemical Reaction Models in Synthetic Promoter Design in Bacteria.

We discuss the formalism of chemical reaction networks (CRNs) as a computer-aided design interface for using formal methods in engineering living technologies. We set out by reviewing formal methods within a broader view of synthetic biology. Based on published results, we illustrate, step by step, how mathematical and computational techniques on CRNs can be used to study the structural and dynamic properties of the designed systems. As a case study, we use an E. coli two-component system that relays the external inorganic phosphate concentration signal to genetic components. We show how CRN models can scan and explore phenotypic regimes of synthetic promoters with varying detection thresholds, thereby providing a means for fine-tuning the promoter strength to match the specification.

Promoter Prediction in Agrobacterium tumefaciens Strain C58 by Using Artificial Intelligence Strategies.

Promoters are the genomic regions upstream of genes that RNA polymerase binds in order to initiate gene transcription. Understanding the regulation of gene expression depends on being able to identify promoters, because they are the most important component of gene expression. Agrobacterium tumefaciens (A. tumefaciens) strain C58 was the subject of this study with the goal of creating a machine learning-based model to predict promoters. In this study, nucleotide density (ND), k-mer, and one-hot were used to encode the promoter sequence. Support vector machine (SVM) on fivefold cross-validation with incremental feature selection (IFS) was used to optimize the generated features. These improved characteristics were then used to distinguish promoter sequences by feeding them into the random forest (RF) classifier. Tenfold cross-validation (CV) analysis revealed that the projected model has the ability to produce an accuracy of 84.22%.

Promoters and Synthetic Promoters in Trichoderma reesei.

Trichoderma reesei holds immense promise for large-scale protein production, rendering it an excellent subject for deeper exploration using genetic engineering methods to achieve a comprehensive grasp of its cellular physiology. Understanding the genetic factors governing its intrinsic regulatory network is crucial, as lacking this knowledge could impede the expression of target genes. Prior and ongoing studies have concentrated on advancing new expression systems grounded in synthetic biology principles. These methodologies involve utilizing established potent promoters or engineered variations. Genomic and transcriptomic analyses have played a pivotal role in identifying robust promoters and expression systems, including light-responsive, copper-inducible, L-methionine-inducible, and Tet-On systems, among others. This chapter seeks to highlight various research endeavors focusing on tunable and constitutive promoters, the impact of different promoters on both native and foreign protein expression, the discovery of fresh promoters, and strategies conducive to future research aimed at refining and enhancing protein expression in T. reesei. Characterizing new promoters and adopting innovative expression systems hold the potential to significantly expand the molecular toolkit accessible for genetically engineering T. reesei strains. For instance, modifying potent inducible promoters such as Pcbh1 by replacing transcriptional repressors (cre1, ace1) with activators (xyr1, ace2, ace3, hap2/3/5) and integrating synthetic expression systems can result in increased production of crucial enzymes such as endoglucanases (EGLs), ß-glucosidases (BGLs), and cellobiohydrolases (CBHs). Similarly, robust constitutive promoters such as Pcdna1 can be converted into synthetic hybrid promoters by incorporating activation elements from potent inducible promoters, facilitating cellulase induction and expression even under repressive conditions. Nevertheless, further efforts are necessary to uncover innovative promoters and devise novel expression strategies to enhance the production of desired proteins on an industrial scale.

Hybrid Synthetic Promoters in Saccharomyces cerevisiae Built on Foreign Promoter Sequences.

Traditionally, hybrid promoters are constructed, in Saccharomyces cerevisiae, by joining the core region and the upstream activating sequences from different native promoters. Here, we describe a new design that makes use of the core promoters from foreign organisms: viruses, humans, and the yeast Schizosaccharomyces pombe. With this approach, we realized a library of 59 new constitutive promoters that span over nine folds in gene expression.

Promoters in Pichia pastoris: A Toolbox for Fine-Tuned Gene Expression.

This chapter reviews the different promoters used to control gene expression in the yeast Pichia pastoris, mainly for recombinant protein production. It covers natural inducible, derepressed, and constitutive promoters, as well as engineered synthetic/hybrid promoters, orthologous promoters from related yeasts, and emerging bidirectional promoters. Key examples, characteristics, and regulatory mechanisms are discussed for each promoter class. Recent efforts in promoter engineering through rational design, mutagenesis, and computational approaches are also highlighted. Looking ahead, we anticipate further developments that will enhance promoter design for Pichia pastoris. Overall, this comprehensive overview underscores the importance of promoter choice and engineering for fully harnessing Pichia pastoris biotechnological potential.

Reliable Genomic Integration Sites in Pseudomonas putida Identified by Two-Dimensional Transcriptome Analysis.

Genomic integration is commonly used to engineer stable production hosts. However, so far, for many microbial workhorses, only a few integration sites have been characterized, thereby restraining advanced strain engineering that requires multiple insertions. Here, we report on the identification of novel genomic integration sites, so-called landing pads, for Pseudomonas putida KT2440. We identified genomic regions with constant expression patterns under diverse experimental conditions by using RNA-Seq data. Homologous recombination constructs were designed to insert heterologous genes into intergenic sites in these regions, allowing condition-independent gene expression. Ten potential landing pads were characterized using four different msfGFP expression cassettes. An insulated probe sensor was used to study locus-dependent effects on recombinant gene expression, excluding genomic read-through of flanking promoters under changing cultivation conditions. While the reproducibility of expression in the landing pads was very high, the msfGFP signals varied strongly between the different landing pads, confirming a strong influence of the genomic context. To showcase that the identified landing pads are also suitable candidates for heterologous gene expression in other Pseudomonads, four equivalent landing pads were identified and characterized in Pseudomonas taiwanensis VLB120. This study shows that genomic "hot" and "cold" spots exist, causing strong promoter-independent variations in gene expression. This highlights that the genomic context is an additional parameter to consider when designing integrable genomic cassettes for tailored heterologous expression. The set of characterized genomic landing pads presented here further increases the genetic toolbox for deep metabolic engineering in Pseudomonads.

Bioinformatic Prediction and High Throughput In Vivo Screening to Identify Cis-Regulatory Elements for the Development of Algal Synthetic Promoters.

Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, their development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity. Combining the algorithms POWRS, STREME, and PhyloGibbs, we predicted 1511 CREs and inserted them into a minimal synthetic promoter sequence in 1, 2, or 3 copies, resulting in 4533 distinct synthetic promoters. These promoters were evaluated in vivo for their capacity to drive the expression of a transgene in a high-throughput manner through next-generation sequencing post antibiotic selection and fluorescence-activated cell sorting. To validate our approach, we sequenced hundreds of transgenic lines showing high levels of GFP expression. Further, we individually tested 14 identified promoters, revealing substantial increases in GFP expression─up to nine times higher than the baseline synthetic promoter, with five matching or even surpassing the performance of the native AR1 promoter. As a result of this study, we identified a catalog of CREs that can now be used to build superior synthetic algal promoters. More importantly, here we present a validated pipeline to generate building blocks for innovative synthetic genetic tools applicable to any algal species with a sequenced genome and transcriptome data set.

In vitro conjugation of IncB/O-plasmid: Minimum inhibitory concentration of ß-lactams increases 16-fold in Salmonella enterica compared with Escherichia coli.

The use of antimicrobials in poultry leaves residues in the litter, favoring the emergence of antimicrobial-resistant pathogens and making it a source of contamination. An in vitro 4 × 4 factorial trial was performed to investigate the influence of four treatments, consisting of antimicrobial sub-concentrations, on the transference of IncB/O-plasmid through conjugation in four groups. Each group was composed of one plasmid donor bacterium (Escherichia coli H2332) and a recipient bacterium (Escherichia coli J62 or Salmonella enterica serovars, Enteritidis, Typhimurium, or Heidelberg). Our results showed a little decrease in the conjugation frequency in almost all treatments between the two bacterial species, which varied according to each strain. The MIC test revealed an increase of up to 4096-fold in resistance to beta-lactams in Salmonella serovars after plasmid acquisition. This finding suggests that some genetic apparatus may be involved in increased antimicrobial resistance in Salmonella serovars after the acquisition of primary resistance determinants.