Quorum-sensing (QS) systems based on acyl-homoserine lactones (AHLs) regulate gene expression in response to cell density in many bacteria, including Rhizobium. These systems, typically composed of LuxI-like synthases and LuxR-like regulators, control processes such as plasmid conjugation, biofilm formation, and plant interactions. However, their evolutionary dynamics and genomic distribution in Rhizobium remain poorly understood. We analyzed 142 complete Rhizobium genomes using comparative genomics, phylogenetic reconstruction, and genomic context analysis. LuxI/LuxR homologs were identified based on sequence similarity and Pfam domain architecture, and their genomic contexts were examined. Phylogenetic relationships and coevolution between LuxI/LuxR pairs were assessed using cophylogenetic approaches. QS systems showed a highly heterogeneous distribution across Rhizobium genomes: some strains lacked canonical systems, whereas others encoded one or multiple systems in chromosomes and/or plasmids. Chromosomal QS systems were associated with multiple distinct genomic contexts, supporting at least seven independent acquisition events. In contrast, plasmid-encoded systems exhibited substantially greater diversity in both sequence and genomic organization. Phylogenetic and comparative analyses revealed dynamic gains and losses of QS systems, variable coevolution among LuxI/LuxR pairs, and evidence of partner recruitment. Notably, plasmids appear to act as major reservoirs of QS systems and likely sources of their transfer to chromosomes. These findings indicate that QS systems in Rhizobium evolve through a combination of horizontal gene transfer, genomic rearrangement, and differential retention across replicons. The higher diversity and mobility of plasmid-encoded systems highlight their central role in shaping QS evolution and functional innovation. Overall, this study provides a comprehensive framework for understanding the diversification and evolutionary trajectories of QS systems in complex multipartite bacterial genomes.

Escherichia coli is one of the leading microbial chassis for bioproduct manufacturing in biotechnology. However, its use in high-temperature bioprocesses is restricted to its mesophilic nature. Rational engineering for improving thermotolerance in E. coli is challenging due to the limited understanding of the molecular functions and interactions involved in supraoptimal thermal adaptation. In recent decades, various approaches have been applied to increase the thermotolerance of E. coli. In this review, we examine the effect of temperature on cellular growth and thermal adaptation at supraoptimal temperatures and discuss how this knowledge can be applied to increase thermotolerance in E. coli. We particularly emphasize systems, synthetic, and evolutionary biology approaches that translate into systems metabolic engineering strategies to improve E. coli thermotolerance. We expect that systems-level insights into heat-stress physiology will enable data-driven strategies for the development of thermotolerant E. coli strains that can be used in high-temperature bioprocesses.

Improving Escherichia coli’s thermotolerance through rational engineering is hindered by limited knowledge of the molecular mechanisms involved in supraoptimal thermal adaptation. To address this issue, we applied a reverse metabolic engineering strategy to develop a d-homolactic E. coli strain under non-aerated conditions. We first characterized the thermal reaction norms of the kinetic and stoichiometric parameters in strains evolved through continuous adaptive laboratory evolution, identifying those that maintained parental volumetric productivity and the lactate/glucose yield at high temperatures. Then, the genomic analysis of two thermally adapted strains was performed to determine the mutations acquired during thermal adaptation. Thermally adapted strains revealed convergent mutations in regulatory genes, notably metJ, lrp, and components of the RNA polymerase complex. Introducing these point mutations into the parental strain demonstrated that individual mutations in rpoB and metJ significantly improved growth at high temperatures, despite the presence of complex epistatic interactions in combinatorial analyses. • ALE significantly enhanced the thermotolerance of homolactic Escherichia coli • Mutations in rpoB and metJ notably enhance growth at high temperatures • Lactate productivity and yield stayed similar to the parental strain

Notwithstanding the ecological significance of the Cladosporium genus, which includes several cosmopolitan fungi reported as saprobes, plant pathogens, and causes of allergic and cutaneous diseases in humans, only 41 genomes have been reported. Here, we report the genome of a polyurethane (PU)-degrading fungus initially identified as Cladosporium tenuissimum A3.I.1. PU is a plastic material widely used as mattress foams, building insulation, protective sealants, and fabrics, that is highly resistant to degradation and barely recycled, hence its waste remains in the environment for hundreds of years. After sequencing its genome using three different techniques-Illumina, PacBio SMRT RSII, and Hi-C-and performing different bioinformatic analyses, the A3.I.1 species placement was reassessed. This analysis provides the first high-quality chromosome-level genome assembly, with 33,552,215 bp, a genome coverage of 58.7X, a contig N50 of 1,881,040 bp, and a GC content of 53%. Assembly scaffolding with Proximo Hi-C revealed 19 scaffolds, corresponding to the total number of haploid chromosomes. The phylophenetic analysis supports its re-classification as Cladosporium oxysporum strain A3.I.1, refining its taxonomic placement. When comparing A3.I.1 against the reference genome of C. oxysporum strain AAAS_A1, 693,670 single-nucleotide variants, 18,623 insertions, and 18,687 deletions were uncovered in the A3.I.1 genome. Gene prediction using FunGAP identified 13,115 protein-coding genes, 121 tRNA genes, and 69 rRNA genes. This genomic resource provides valuable insights into environmental resilience research and supports the emerging role of C. oxysporum A3.I.1 as a PU degrader. It also opens new venues for the development of sustainable biorecycling processes.

Species outside the Acinetobacter calcoaceticus–baumannii (Acb) complex have recently emerged as new clinically relevant carbapenem-resistant pathogens, such as A. haemolyticus. A new mechanism that could contribute to the dissemination of this resistance is the secretion of outer membrane vesicles (OMVs). We investigate OMVs secreted by A. haemolyticus AN54, a Mexican clinical strain carrying a plasmid-borne blaNDM−1 gene and by its plasmid-cured derivative AN54Δe. Active secretion of OMVs formed by a lipid membrane, measuring 10–50 nm in both strains, was observed by TEM. The OMVs from AN54 possess hydrolytic capacity against carbapenem. The OMV protein profile varied depending on the antibiotic concentration during induction, with additional protein bands detected at 8 and 32 µg/ml of antibiotic exposure. Mass spectrometry identified proteins involved in different metabolic pathways and in resistance-related processes. The porins, efflux pumps, and ADC enzyme were more abundant in the OMVs from AN54Δe than in those from AN54 harboring the active NDM-1 enzyme. The OMVs protected the sensitive strains from the same and different genera against the antibiotic action for a limited period. However, the OMVs transferred the blaNDM−1-carrying plasmid (pAhaeAN54e) only to the AN54Δe strain, conferring permanent resistance to carbapenems. Together, these findings suggest that OMVs can help protect surrounding bacteria from antibiotic action and serve as vehicles for disseminating resistance genes. These results show the importance of studying OMVs as a novel mechanism for the dissemination of antimicrobial resistance.

Emerging diseases caused by fungi are a serious threat to wildlife biodiversity. The widespread fungal pathogen Batrachochytrium dendrobatidis (Bd) has caused dramatic amphibian population declines and species extinctions worldwide. While many amphibians have been negatively affected by Bd, some populations/species have persisted despite its presence. One factor contributing to amphibian protection against this fungus is the host-associated skin microbiome. In this study, we aimed to identify gene clusters associated with the antifungal activity of amphibian skin bacteria. Specifically, we explored skin bacteria isolated from species that have persisted in the wild despite the presence of Bd: the frogs Agalychnis callidryas, Craugastor fitzingeri, Dendropsophus ebraccatus, and the axolotl Ambystoma altamirani. Bacterial isolates were tested in vitro for their capacity to inhibit the growth of two fungal pathogens: Bd and the phytopathogen Botrytis cinerea (Bc). Genome mining of these bacterial isolates revealed a diverse repertoire of Biosynthetic Gene Clusters (BGCs) and chitin-degrading gene families (ChDGFs) whose composition and abundance differed among bacterial families. We found specific BGCs and ChDGFs that were associated with the capacity of bacteria to inhibit the growth of either Bd or Bc, suggesting that distinct fungi could be inhibited by different molecular mechanisms. By using similarity networks and machine learning, we identified BGCs encoding known antifungal compounds such as viscosin, fengycin, zwittermicin, as well as siderophores and a novel family of beta-lactones. Finally, we propose that the diversity of BGCs found in amphibian skin bacteria comprise a substantial genetic reservoir that could collectively explain the antifungal properties of the amphibian skin microbiome.

Here, we report the characterization of two Cupriavidus strains, NOV2-1 and OV2-1, isolated from an iron-oxide deposit in an underground tunnel of the Naica mine in Mexico. This unique biotope, characterized by its high temperature (≈50 °C) and the presence of heavy metals, is no longer available for sampling at this time. The genomes of NOV2-1 and OV2-1 comprised two replicons: a chromosome of 3.58 and 3.53 Mb, respectively, and a chromid of 2.1 Mb in both strains. No plasmids were found. The average nucleotide identity and the core genome phylogeny showed that NOV2-1 and OV2-1 belonged to the Cupriavidus gilardii species. NOV2-1 and OV2-1 grew up to 48 °C, with an optimal temperature of 42 °C. Discrete differences were observed between C. gilardii CCUG38401T, NOV2-1, and OV2-1 in the biochemical tests. NOV2-1 and OV2-1 presented resistance to zinc, lead, copper, cadmium, nickel, and cobalt. Several complete and incomplete gene clusters related to the resistance to these heavy metals (ars, czc, cop 1, sil-cop 2, cup, mmf, and mer) were detected in the genome of these strains. Although further studies are needed to determine the origin and role of the detected gene clusters, it is suggested that the czc system may have been mobilized by horizontal gene transfer. This study expands the extreme biotopes where Cupriavidus strains can be retrieved.

The heat shock response is a cellular protection mechanism against sudden temperature upshifts extensively studied in Escherichia coli. However, the effects of thermal evolution on this response remain largely unknown. In this study, we investigated the early and late physiological and transcriptional responses to temperature upshift in a thermotolerant strain under continuous culture conditions. Adaptive laboratory evolution was performed on a metabolically engineered E. coli strain (JU15), designed for d-lactic acid production, to enable cellular growth and fermentation of glucose at 45 °C in batch cultures. The resulting homofermentative strain, ECL45, successfully adapted to 45 °C in a glucose-mineral medium at pH 7 under non-aerated conditions. The thermal-adapted ECL45 retained the parental strain’s high volumetric productivity and product/substrate yield. Genomic sequencing of ECL45 revealed eight mutations, including one in a non-coding region and six within the coding regions of genes associated with metabolic, transport, and regulatory functions. Transcriptomic analysis comparing the evolved strain with its parental counterpart under early and late temperature upshifts indicated that the adaptation involved a controlled stringent response. This mechanism likely contributes to the strain’s ability to maintain growth capacity at high temperatures. • The temperature upshift response of a thermally adapted strain in continuous culture was studied for the first time. • Genomic analyses revealed the presence of a double point mutation in the spoT gene. • The thermally adapted strain maintained underexpression of the spoT gene at high temperatures. • Supplementation of 0.15 g/L of hydrolyzed protein favored thermal adaptation at 45 °C.

Acinetobacter baumannii is a major nosocomial pathogen worldwide and, specifically, in Latin America. Genomic epidemiology has been instrumental in determining the transmission dynamics of A. baumannii in many countries of the world, yet some Latin American countries have conducted no genomic epidemiology studies. Here, we conduct the first genomic epidemiology study about this pathogen in Paraguay. We sequenced 43 isolates from a big tertiary hospital in Paraguay collected from different wards in 2021 and 2022. Our genomic epidemiology analyses, including almost 200 genomes and considering the main international clones (ICs), show that IC1, IC2, IC4, IC5, and IC7 were found in the hospital. We found novel genetic variation (three novel sequence types as per the Oxford MLST scheme and one as per the Pasteur scheme) within IC7. Antibiotic susceptibility tests show that all but one of the Paraguayan isolates were resistant to carbapenems. Notably, 98% were classified as multidrug-resistant. We detected plasmids in almost all the Paraguayan isolates. Furthermore, we detected cases of recent horizontal transfer of important antibiotic resistance genes between different ICs. On a general note, our findings highlight polyclonal spreading across different hospital wards and horizontal transfer of clinically relevant antibiotic resistance genes among the different clones. On a more local note, this is the first genomic epidemiology study of A. baumannii in Paraguay and will be a reference point for future studies in the country and the region.

Acinetobacter baumannii is a major nosocomial pathogen worldwide and, specifically, in Latin America. Genomic epidemiology has been instrumental in determining the transmission dynamics of A. baumannii in many countries of the world, yet some Latin American countries have conducted no genomic epidemiology studies. Here, we conduct the first genomic epidemiology study about this pathogen in Paraguay. We sequenced 43 isolates from a big tertiary hospital in Paraguay collected from different wards in 2021 and 2022. Our genomic epidemiology analyses, including almost 200 genomes and considering the main international clones (ICs), show that IC1, IC2, IC4, IC5, and IC7 were found in the hospital. We found novel genetic variation (three novel sequence types as per the Oxford MLST scheme and one as per the Pasteur scheme) within IC7. Antibiotic susceptibility tests show that all but one of the Paraguayan isolates were resistant to carbapenems. Notably, 98% were classified as multidrug-resistant. We detected plasmids in almost all the Paraguayan isolates. Furthermore, we detected cases of recent horizontal transfer of important antibiotic resistance genes between different ICs. On a general note, our findings highlight polyclonal spreading across different hospital wards and horizontal transfer of clinically relevant antibiotic resistance genes among the different clones. On a more local note, this is the first genomic epidemiology study of A. baumannii in Paraguay and will be a reference point for future studies in the country and the region.

Plants and microorganisms establish beneficial associations that can improve their development and growth. Recently, it has been demonstrated that bacteria isolated from the skin of amphibians can contribute to plant growth and defense. However, the molecular mechanisms involved in the beneficial effect for the host are still unclear. In this work, we explored whether bacteria isolated from three tropical frogs species can contribute to plant growth. After a wide screening, we identified three bacterial strains with high biostimulant potential, capable of modifying the root structure of Arabidopsis thaliana plants. In addition, applying individual bacterial cultures to Solanum lycopersicum plants induced an increase in their growth. To understand the effect that these microorganisms have over the host plant, we analysed the transcriptomic profile of A. thaliana during the interaction with the C32I bacterium, demonstrating that the presence of the bacteria elicits a transcriptional response associated to plant hormone biosynthesis. Our results show that amphibian skin bacteria can function as biostimulants to improve agricultural crops growth and development by modifying the plant transcriptomic responses.

Introduction:The Acinetobacter calcoaceticus-Acinetobacter baumannii complex, or Acb complex, consists of six species: Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii, and Acinetobacter lactucae. A. baumannii is the most clinically significant of these species and is frequently related to healthcare-associated infections (HCAIs). Clustered regularly interspaced short palindromic repeat (CRISPR) arrays and associated genes (cas) constitute bacterial adaptive immune systems and function as variable genetic elements. This study aimed to conduct a genomic analysis of Acb complex genomes available in databases to describe and characterize CRISPR systems and cas genes. Methods:Acb complex genomes available in the NCBI and BV-BRC databases, the identification and characterization of CRISPR-Cas systems were performed using CRISPRCasFinder, CRISPRminer, and CRISPRDetect. Sequence types (STs) were determined using the Oxford scheme and ribosomal multilocus sequence typing (rMLST). Prophages were identified using PHASTER and Prophage Hunter. Results:A total of 293 genomes representing six Acb species exhibited CRISPR-related sequences. These genomes originate from various sources, including clinical specimens, animals, medical devices, and environmental samples. Sequence typing identified 145 ribosomal multilocus sequence types (rSTs). CRISPR-Cas systems were confirmed in 26.3% of the genomes, classified as subtypes I-Fa, I-Fb and I-Fv. Probable CRISPR arrays and cas genes associated with CRISPR-Cas subtypes III-A, I-B, and III-B were also detected. Some of the CRISPR-Cas systems are associated with genomic regions related to Cap4 proteins, and toxin-antitoxin systems. Moreover, prophage sequences were prevalent in 68.9% of the genomes. Analysis revealed a connection between these prophages and CRISPR-Cas systems, indicating an ongoing arms race between the bacteria and their bacteriophages. Furthermore, proteins associated with anti-CRISPR systems, such as AcrF11 and AcrF7, were identified in the A. baumannii and A. pittii genomes. Discussion:This study elucidates CRISPR-Cas systems and defense mechanisms within the Acb complex, highlighting their diverse distribution and interactions with prophages and other genetic elements. This study also provides valuable insights into the evolution and adaptation of these microorganisms in various environments and clinical settings.

Botrytis cinerea is the causal agent of gray mold, which affects a wide variety of plant species. Chemical agents have been used to prevent the disease caused by this pathogenic fungus. However, their toxicity and reduced efficacy have encouraged the development of new biological control alternatives. Recent studies have shown that bacteria isolated from amphibian skin display antifungal activity against plant pathogens. However, the mechanisms by which these bacteria act to reduce the effects of B. cinerea are still unclear. From a diverse collection of amphibian skin bacteria, three proved effective in inhibiting the development of B. cinerea under in vitro conditions. Additionally, the individual application of each bacterium on the model plant Arabidopsis thaliana, Solanum lycopersicum and post-harvest blueberries significantly reduced the disease caused by B. cinerea. To understand the effect of bacteria on the host plant, we analyzed the transcriptomic profile of A. thaliana in the presence of the bacterium C32I and the fungus B. cinerea, revealing transcriptional regulation of defense-related hormonal pathways. Our study shows that bacteria from the amphibian skin can counteract the activity of B. cinerea by regulating the plant transcriptional responses.

We report the draft genomes of four Kluyveromyces marxianus isolates obtained from the elaboration process of henequen (Agave fourcroydes) mezcal, a Mexican alcoholic beverage. The average nucleotide identity analysis revealed that isolates derived from agave plants are distinct from those from other environments, including agave fermentations.

The blaNDM-1 gene and its variants encode metallo-beta-lactamases that confer resistance to almost all beta-lactam antibiotics. Genes encoding blaNDM-1 and its variants can be found in several Acinetobacter species, and they are usually linked to two different plasmid clades. The plasmids in one of these clades contain a gene encoding a Rep protein of the Rep_3 superfamily. The other clade consists of medium-sized plasmids in which the gene (s) involved in plasmid replication initiation (rep)have not yet been identified. In the present study, we identified the minimal replication region of a blaNDM-1-carrying plasmid of Acinetobacter haemolyticus AN54 (pAhaeAN54e), a member of this second clade. This region of 834 paired bases encodes three small peptides, all of which have roles in plasmid maintenance. The plasmids containing this minimal replication region are closely related; almost all contain blaNDM genes, and they are found in multiple Acinetobacter species, including A. baumannii. None of these plasmids contain an annotated Rep gene, suggesting that their replication relies on the minimal replication region that they share with the plasmid pAhaeAN54e. These observations suggest that this plasmid lineage plays a crucial role in the dissemination of the blaNDM-1 gene and its variants.

In this study, we examined the role of the lipopolysaccharide (LPS) core of Rhizobium etli in facilitating the adsorption and infection of phages with broad host range. When the plasmid-encoded LPS biosynthesis genes, wreU and wreV, were disrupted, distinct and contrasting effects on phage infection were observed. The wreU mutant strains exhibited wild-type adsorption and infection properties, whereas the wreV mutant demonstrated resistance to phage infection, but retained the capacity to adsorb phages. Complementation of the wreV mutant strains with a recombinant plasmid containing the wreU and wreV, restored the susceptibility to the phages. However, the presence of this recombinant plasmid in a strain devoid of the native lps-encoding plasmid was insufficient to restore phage susceptibility. These results suggest that the absence of wreV impedes the proper assembly of the complete LPS core, potentially affecting the formation of UDP-KdgNAg or KDO precursors for the O-antigen. In addition, a protein not yet identified, but residing in the native lps-encoding plasmid, may be necessary for complete phage infection.

Acinetobacter pittii has increasingly been associated with several types of hospital-acquired severe infections. Genes implicated in carbapenem resistance, tigecycline resistance, or genes encoding extended spectrum cephalosporinases, such as blaADC, are commonly found in isolates implicated in these infections. A. pittii strains that are pandrug resistant have occasionally been identified. Food for human consumption, animals and plants are environmental sources of this pathogen. An alarming situation is that A. pitti has been identified as responsible for outbreaks in different regions worldwide. In this study, 384 genomes of A. pittii were analyzed, comprising sequences from clinical and non-clinical origins from 32 countries. The objective was to investigate if clinical strains possess genetic traits facilitating hospital adaptation. Results indicate significant genomic variability in terms of size and gene content among A. pittii isolates. The core genome represents a small portion (25–36%) of each isolate’s genome, while genes associated with antibiotic resistance and virulence predominantly belong to the accessory genome. Notably, antibiotic resistance genes are encoded by a diverse array of plasmids. As the core genome between environmental and hospital isolates is the same, we can assume that hospital isolates acquired ARGs due to a high selective pressure in these settings. The strain’s phylogeographic distribution indicates that there is no geographical bias in the isolate distribution; isolates from different geographic regions are dispersed throughout a core genome phylogenetic tree. A single clade may include isolates from extremely distant geographical areas. Furthermore, strains isolated from the environment or animal, or plant sources frequently share the same clade as hospital isolates. Our analysis showed that the clinical isolates do not already possess specific genes, other than antibiotic-resistant genes, to thrive in the hospital setting.

Acinetobacter pittii 978-A_19 was obtained from a parrot with pneumonia. It is resistant to ampicillin, carbenicillin, cephalosporins, clindamycin, and trimethoprim + sulfamethoxazole. The genome encodes a new blaADC allele, a blaOXA-502 gene, possesses several virulence genes related to adherence and biofilm formation, and has types I, II, and IV secretion systems.

Introduction: The ζ subunit is a potent inhibitor of the F1FO-ATPase of Paracoccus denitrificans (PdF1FO-ATPase) and related α-proteobacteria different from the other two canonical inhibitors of bacterial (ε) and mitochondrial (IF1) F1FO-ATPases. ζ mimics mitochondrial IF1 in its inhibitory N-terminus, blocking the PdF1FO-ATPase activity as a unidirectional pawl-ratchet and allowing the PdF1FO-ATP synthase turnover. ζ is essential for the respiratory growth of P. denitrificans, as we showed by a Δζ knockout. Given the vital role of ζ in the physiology of P. denitrificans, here, we assessed the evolution of ζ across the α-proteobacteria class. Methods: Through bioinformatic, biochemical, molecular biology, functional, and structural analyses of several ζ subunits, we confirmed the conservation of the inhibitory N-terminus of ζ and its divergence toward its C-terminus. We reconstituted homologously or heterologously the recombinant ζ subunits from several α-proteobacteria into the respective F-ATPases, including free-living photosynthetic, facultative symbiont, and intracellular facultative or obligate parasitic α-proteobacteria. Results and discussion: The results show that ζ evolved, preserving its inhibitory function in free-living α-proteobacteria exposed to broad environmental changes that could compromise the cellular ATP pools. However, the ζ inhibitory function was diminished or lost in some symbiotic α-proteobacteria where ζ is non-essential given the possible exchange of nutrients and ATP from hosts. Accordingly, the ζ gene is absent in some strictly parasitic pathogenic Rickettsiales, which may obtain ATP from the parasitized hosts. We also resolved the NMR structure of the ζ subunit of Sinorhizobium meliloti (Sm-ζ) and compared it with its structure modeled in AlphaFold. We found a transition from a compact ordered non-inhibitory conformation into an extended α-helical inhibitory N-terminus conformation, thus explaining why the Sm-ζ cannot exert homologous inhibition. However, it is still able to inhibit the PdF1FO-ATPase heterologously. Together with the loss of the inhibitory function of α-proteobacterial ε, the data confirm that the primary inhibitory function of the α-proteobacterial F1FO-ATPase was transferred from ε to ζ and that ζ, ε, and IF1 evolved by convergent evolution. Some key evolutionary implications on the endosymbiotic origin of mitochondria, as most likely derived from α-proteobacteria, are also discussed.

The CS21 pilus produced by enterotoxigenic Escherichia coli (ETEC) is involved in adherence to HT-29 intestinal cells. The CS21 pilus assembles proteins encoded by 14 genes clustered into the lng operon. Aim. This study aimed to determine whether E. coli BL21 (ECBL) transformed with the lng operon lacking the lngA gene (pE9034AΔlngA) and complemented in trans with lngA variants of ETEC clinical strains, as well as point substitutions, exhibited modified adherence to HT-29 cells. Methods. A kanamycin cassette was used to replace the lngA gene in the lng operon of the E9034A strain, and the construct was transformed into the ECBL strain. The pJET1.2 vector carrying lngA genes with allelic variants was transformed into ECBLpE9034AΔlngA (ECBLΔlngA). The point substitutions were performed in the pJETlngAFMU073332 vector. Results. Bioinformatic alignment analysis of the LngA proteins showed hypervariable regions and clustered the clinical ETEC strains into three groups. Variations in amino acid residues affect the adherence percentages of recombinant ECBL strains with lngA variants and site-specific mutations with HT-29 cells. Conclusion. In this study, ECBL carrying the lng operon harboring lngA variants of six clinical ETEC strains, as well as point substitutions, exerted an effect on the adherence of ECBL to HT-29 cells, thereby confirming the importance of the CS21 pilus in adherence.