Frequent Horizontal Gene Transfer Research Articles

Tire wear particles enhance horizontal gene transfer of antibiotic resistance genes in aquatic ecosystems

Microplastics (MPs) have introduced new surfaces for biofilm development and gene exchange among bacteria. We investigated Tire Wear Particles (TWPs) for their involvement in horizontal gene transfer (HGT), particularly in relation to associated metals in the matrices of TWPs. We employed red-fluorescently tagged E. coli strain as a donor with green-fluorescently tagged, broad-host-range plasmid pKJK5, resistant to trimethoprim. As a recipient, we utilized Pseudomonas sp. and a natural lake microbial community. HGT activity on TWPs was determined and compared with that on polystyrene (PS) (with and without metals), and chitosan, which was used as a natural surface. Exposure to TWPs significantly enhanced HGT frequency of antibiotic resistance gene (ARG) from donor to recipient compared to PS and chitosan, and metals of TWPs further promoted HGT. HGT frequency on TWPs with Pseudomonas sp. was found to be 10−3 at 30 °C. in the lake community, it was similarly high already at 25 °C suggesting a higher permissiveness of the natural microbial community towards ARG at lower temperatures. This study sheds light on the potential impact of TWPs in promoting HGT, forming the basis for health risk assessments of TWPs and more generally of MP pollution in various aquatic ecosystems.

Evolution of plant metabolism: the state-of-the-art.

Immense chemical diversity is one of the hallmark features of plants. This chemo-diversity is mainly underpinned by a highly complex and biodiverse biochemical machinery. Plant metabolic enzymes originated and were inherited from their eukaryotic and prokaryotic ancestors and further diversified by the unprecedentedly high rates of gene duplication and functionalization experienced in land plants. Unlike prokaryotic microbes, which display frequent horizontal gene transfer events and multiple inputs of energy and organic carbon, land plants predominantly rely on organic carbon generated from CO2 and have experienced relatively few gene transfers during their recent evolutionary history. As such, plant metabolic networks have evolved in a stepwise manner using existing networks as a starting point and under various evolutionary constraints. That said, until recently, the evolution of only a handful of metabolic traits had been extensively investigated and as such, the evolution of metabolism has received a fraction of the attention of, the evolution of development, for example. Advances in metabolomics and next-generation sequencing have, however, recently led to a deeper understanding of how a wide range of plant primary and specialized (secondary) metabolic pathways have evolved both as a consequence of natural selection and of domestication and crop improvement processes. This article is part of the theme issue 'The evolution of plant metabolism'.

High-throughput single-cell sequencing of activated sludge microbiome

Wastewater treatment plants (WWTPs) represent one of biotechnology's largest and most critical applications, playing a pivotal role in environmental protection and public health. In WWTPs, activated sludge (AS) plays a major role in removing contaminants and pathogens from wastewater. While metagenomics has advanced our understanding of microbial communities, it still faces challenges in revealing the genomic heterogeneity of cells, uncovering the microbial dark matter, and establishing precise links between genetic elements and their host cells as a bulk method. These issues could be largely resolved by single-cell sequencing, which can offer unprecedented resolution to show the unique genetic information. Here we show the high-throughput single-cell sequencing to the AS microbiome. The single-amplified genomes (SAGs) of 15,110 individual cells were clustered into 2,454 SAG bins. We find that 27.5% of the genomes in the AS microbial community represent potential novel species, highlighting the presence of microbial dark matter. Furthermore, we identified 1,137 antibiotic resistance genes (ARGs), 10,450 plasmid fragments, and 1,343 phage contigs, with shared plasmid and phage groups broadly distributed among hosts, indicating a high frequency of horizontal gene transfer (HGT) within the AS microbiome. Complementary analysis using 1,529 metagenome-assembled genomes from the AS samples allowed for the taxonomic classification of 98 SAG bins, which were previously unclassified. Our study establishes the feasibility of single-cell sequencing in characterizing the AS microbiome, providing novel insights into its ecological dynamics, and deepening our understanding of HGT processes, particularly those involving ARGs. Additionally, this valuable tool could monitor the distribution, spread, and pathogenic hosts of ARGs both within AS environments and between AS and other environments, which will ultimately contribute to developing a health risk evaluation system for diverse environments within a One Health framework.

Exploring the antibiotic resistance genes removal dynamics in chicken manure by composting

Prolonged antibiotic usage in livestock farming leads to the accumulation of antibiotic resistance genes in animal manure. Composting has been shown as an effective way of removing antibiotic resistance from manures, but the specific mechanisms remain unclear. This study used time-series sampling and metagenomics to analyse the resistome types and their bacterial hosts in chicken manures. Composting significantly altered the physicochemical properties and microbiome composition, reduced antibiotic resistance genes by 65.71 %, mobile genetic elements by 68.15 % and horizontal gene transfer frequency. Source tracking revealed that Firmicutes, Actinobacteria, and Proteobacteria are the major bacterial hosts involved in the resistome and gene transfer events. Composting reduces the resistome risk by targeting pathogens such as Staphylococcus aureus. Structural equation modelling confirmed that composting reduces resistome risk by changing pH and pathogen abundance. This study demonstrates that composting is an effective strategy for mitigating resistome risk in chicken manure, thereby supporting the One Health initiative.

Evaluation of the potential horizontal gene transfer ability during chicken manure and pig manure composting

Resistance genes have been identified as emerging pollutants due to their ability to rapidly spread in the environment through horizontal gene transfer (HGT). Microbial community serves as the pivotal factor influencing the frequency of HGT during manure composting. However, the characteristics of HGT in microbial community from different types of manure were unclear. Therefore, this study aimed to evaluate the potential risk of HGT in bacterial community through the co-composting of chicken manure and pig manure in different proportions. The experimental results showed that the abundance of sulfonamide antibiotic resistance genes and integrase genes was higher during pig manure composting than those during chicken manure composting. In addition, the addition of pig manure also increased resistance genes abundance during chicken manure composting. These results suggested that the potential HGT risk was greater during pig manure composting. Furthermore, microbial analysis of co-composting suggested that bacterial community of pig manure was more competitive and adaptable than that of chicken manure. Ultimately, statistical analysis indicated that compared to chicken manure composting, the potential ability of HGT was greater during pig manure composting. This study provided the vital theoretical support and scientific guidance for mitigating the HGT risk during manure composting.

Mariculture waters as yet another hotbed for the creation and transfer of new antibiotic-resistant pathogenome

With the rapid growth of aquaculture globally, large amounts of antibiotics have been used to treat aquatic disease, which may accelerate induction and spread of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in aquaculture environments. Herein, metagenomic and 16S rRNA analyses were used to analyze the potentials and co-occurrence patterns of pathogenome (culturable and unculturable pathogens), antibiotic resistome (ARGs), and mobilome (mobile genetic elements (MGEs)) from mariculture waters near 5000 km coast of South China. Total 207 species of pathogens were identified, with only 10 culturable species. Furthermore, more pathogen species were detected in mariculture waters than those in coastal waters, and mariculture waters were prone to become reservoirs of unculturable pathogens. In addition, 913 subtypes of 21 ARG types were also identified, with multidrug resistance genes as the majority. MGEs including plasmids, integrons, transposons, and insertion sequences were abundantly present in mariculture waters. The co-occurrence network pattern between pathogenome, antibiotic resistome, and mobilome suggested that most of pathogens may be potential multidrug resistant hosts, possibly due to high frequency of horizontal gene transfer. These findings increase our understanding of mariculture waters as reservoirs of antibiotic resistome and mobilome, and as yet another hotbed for creation and transfer of new antibiotic-resistant pathogenome.

Antibiotic resistance genes profile in the surface sediments of typical aquaculture areas across 15 major lakes in China

Aquatic farming is considered as a major source of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) for the natural environment of the lakes. ARB and ARGs in the natural environment have increased quickly because of the human activities. Here, we have profiled the diversity and abundance of ARGs in sediments from the typical aquaculture areas around 15 major lakes in China using PCR and qPCR, and further assessed the risk factor shaping the occurrence and distribution of ARGs. And class 1, 2 and 3 integrons were initially detected by PCR with specific primers. ARGs were widely distributed in the lakes: Weishan Lake and Poyang Lake showed high diversity of ARGs, followed by Dongting Lake, Chao Lake and Tai Lake. Generally, the ARGs in the Middle-Lower Yangtze Plain were more abundant than those in the Qinghai-Tibet Plateau. Tetracycline resistance genes (tet(C), tet(A) &tet(M)) were prominent in sediments, and the next was AmpC β-lactamase gene group BIL/LAT/CMY, and the last was the genes resistance to aminoglycoside (strA-strB). Partial least squares path modeling analysis (PLS-PMA) revealed that livestock had a significant direct effect on the distribution of ARGs in lakes, and population might indirectly influence the profiles of ARGs by affecting the scale of livestock and aquaculture. The detectable rate of class 1, 2 and 3 integrons were 80%, 100% and 46.67%, respectively. The prevalence of integrons might play a key role in promoting more frequent horizontal gene transfer (HGT) events, resulting in the environmental mobilization and dissemination of ARGs between bacteria.

PMAT: an efficient plant mitogenome assembly toolkit using low-coverage HiFi sequencing data.

Complete mitochondrial genomes (mitogenomes) of plants are valuable resources for nucleocytoplasmic interactions, plant evolution, and plant cytoplasmic male sterile line breeding. However, the complete assembly of plant mitogenomes is challenging due to frequent recombination events and horizontal gene transfers. Previous studies have adopted Illumina, PacBio, and Nanopore sequencing data to assemble plant mitogenomes, but the poor assembly completeness, low sequencing accuracy, and high cost limit the sampling capacity. Here, we present an efficient assembly toolkit (PMAT) for de novo assembly of plant mitogenomes using low-coverage HiFi sequencing data. PMAT has been applied to the de novo assembly of 13 broadly representative plant mitogenomes, outperforming existing organelle genome assemblers in terms of assembly accuracy and completeness. By evaluating the assembly of plant mitogenomes from different sequencing data, it was confirmed that PMAT only requires 1× HiFi sequencing data to obtain a complete plant mitogenome. The source code for PMAT is available at https://github.com/bichangwei/PMAT. The developed PMAT toolkit will indeed accelerate the understanding of evolutionary variation and breeding application of plant mitogenomes.

Horizontal gene transfer in eukaryotes: aligning theory with data.

Horizontal gene transfer (HGT), or lateral gene transfer, is the non-sexual movement of genetic information between genomes. It has played a pronounced part in bacterial and archaeal evolution, but its role in eukaryotes is less clear. Behaviours unique to eukaryotic cells - phagocytosis and endosymbiosis - have been proposed to increase the frequency of HGT, but nuclear genomes encode fewer HGTs than bacteria and archaea. Here, I review the existing theory in the context of the growing body of data on HGT in eukaryotes, which suggests that any increased chance of acquiring new genes through phagocytosis and endosymbiosis is offset by a reduced need for these genes in eukaryotes, because selection in most eukaryotes operates on variation not readily generated by HGT.

Effective attenuation of extracellular antibiotic resistance gene risks in wastewater by capacitive deionization

Despite the effective inactivation on antibiotic resistant bacteria, current water and wastewater treatment technologies are often ineffective in removing antibiotic resistance genes (ARGs), which remain active outside cells, known as extracellular ARGs (eARGs). eARGs can persist for a long time and facilitate bacterial transformation in receiving environments, thereby, posing a threat to public health. This study applied capacitive deionization (CDI), as a promising desalination technology, for eARGs removal from wastewater. Results showed that CDI using carbon electrodes was significantly more effective than activated carbon adsorption in removing eARGs, with the removal efficiency and rate constant that are 2.1–11.2 times (p < 0.05) and 2.5–13 times (p < 0.05) higher. In the flow-by mode, CDI achieved the highest removal of eARGs (3.5 log) in just 15 min. Furthermore, CDI treatment significantly decreased the frequency of horizontal gene transfer (HGT) and increased potential for natural degradation, thereby reducing the risk of eARGs propagation. The high electric field (>1000 V/m) applied in CDI induces DNA conformational damage and DNA sequence mutations, which are the main mechanisms for removing eARG. This study will provide technical guidance and theoretical support for the removal of eARG in wastewater.

Nanowire electroporation-induced cell pores on antibiotic-resistant bacteria to promote chlorine permeation for eliminating intracellular antibiotic resistance genes

Conventional chlorination (Cl2) is inefficient to eliminate intracellular antibiotic resistance genes (i-ARGs) of antibiotic-resistant bacteria (ARB) due to the chlorine permeation barrier and oxidative competition of cell wall/membrane. Herein, we proposed an efficient strategy by constructing synergistic nanowire-induced electroporation (DC-EP) and Cl2 (DC-EP/Cl2) with H2-reduced TiO2 nanowire (TiOxNW) electrodes, attempting to promote i-ARG removal via DC-EP-induced cell damage for chlorine permeation and i-ARG leakage. DC-EP/Cl2 (∼3.7 log and ∼ 60.5 J/L/log) exhibited excellent synergistic effects for ARB inactivation (Escherichia coli) and i-ARG removal (tet(W), aac(3)-II, and blaTEM–1), which achieved much larger i-ARG removal and lower energy consumption than the individual 1.5 V-DC-EP (∼0.05 log) and 10 mg/L-Cl2 (∼0.5 log and ∼ 262.0 J/L/log). Analyses of i-ARG/e-ARG abundances, bacterial cell integrity, and cell morphology indicated that DC-EP-induced cell pores provided channels of chlorine permeation, which was accompanied with oxidative enlargement of cell pores for i-ARG leakage and subsequent degradation. DC-EP/Cl2 exhibited 15–90 times lower frequency of horizontal gene transfer than the individual Cl2 disinfection. The TiOxNW-based DC-EP/Cl2 exhibited excellent stability of i-ARG removal (undetectable ARG abundance and ∼ 20.3 J/L/log) for treating 103 CFU/mL ARB-contaminated tap water within 7-day continuous operation, suggesting its great application potential to alleviate ARG dissemination risk for drinking water.

Dynamic evolution of antibiotic resistance genes in plastisphere in the vertical profile of urban rivers

Microplastics (MPs) can vertically transport in the aquatic environment due to their aging and biofouling, forming distinct plastisphere in different water layers. However, even though MPs have been regarded as hotspots for antibiotic resistance genes (ARGs), little is known about the propagation and transfer of ARGs in plastisphere in waters, especially in the vertical profile. Therefore, this study investigated the dynamic responses and evolution of ARGs in different plastisphere distributed vertically in an urbanized river. The biofilm biomass in the polylactic acid (PLA) plastisphere was relatively higher than that in the polyethylene terephthalate (PET), showing depth-decay variations. The ARGs abundance in plastisphere were much higher than that in the surrounding waters, especially for the PLA. In the vertical profiles, the ARGs abundance in the PET plastisphere increased with water depths, while the highest abundance of ARGs in the PLA mostly appeared at intermediate waters. In the temporal dynamic, the ARGs abundance in plastisphere increased and then decreased, which may be dominated by the MP types at the initial periods. After long-term exposure, the influences of water depths seemed to be strengthened, especially in the PET plastisphere. Compared with surface waters, the microbiota attached in plastisphere in deep waters showed high species richness, strong diversity, and complex interactions, which was basically consistent with the changes of nutrient contents in different water layers. These vertical variations in microbiota and nutrients (e.g., nitrogen) may be responsible for the propagation of ARGs in plastisphere in deep waters. The host bacteria for ARGs in plastisphere was also developed as water depth increased, leading to an enrichment of ARGs in deep waters. In addition, the abundance of ARGs in plastisphere in bottom waters was positively correlated with the mobile genetic elements (MGEs) of intI1 and tnpA05, indicative of a frequent horizontal gene transfer of ARGs. Overall, water depth played a critical role in the propagation of ARGs in plastisphere, which should not be ignored in a long time series. This study provides new insights into the dynamic evolution of ARGs propagation in plastisphere under increasing global MPs pollution, especially in the vertical profile.

Comparison of Horizontal blaCTX-M Gene Transfer via Conjugation among Extended Spectrum β-Lactamases Producing Escherichia coli Isolates from Patients with Urinary Tract Infection, Their Animals, and Environment.

The dissemination of the extended spectrum β-lactamases (ESBL) producing E. coli poses a significant public health problem. Understanding the efficiency and frequency of horizontal gene transfer via conjugation of ESBL producing E. coli is imperative towards devising prevention and control measures. This study compared the frequencies and efficiencies of horizontal blaCTX-M gene transfer via conjugation among Escherichia coli isolates from urine and gastrointestinal tract (GIT) of patients with urinary tract infection (UTI), their animals and environment. Horizontal blaCTX-M gene transfer via conjugation by a broth mating experiment was performed using 50 confirmed ESBL producing E. coli isolates as donors and Escherichia coli J53 (F-, met, pro, Azr), as the recipient. The transconjugants were detected and their frequencies and efficiencies of conjugation were measured and compared between ESBL producing E. coli isolates multi-sourced from urine, GIT, animals and environment. Antimicrobial susceptibility testing of all resulting transconjugants was performed. DNA was extracted from all transconjugants to confirm the presence and the acquisition of blaCTX-M gene. Out of 50 ESBL producing E. coli isolates harboring blaCTX-M gene, 37 (74.0%) successfully exercised horizontal gene transfer through conjugation. All transconjugants were confirmed phenotypically and genotypically by PCR. Of note, all of the isolates from environment 100.0% (7/7) performed conjugation, exhibiting the highest transfer efficiency, followed by isolates from urine and animals, with the conjugation transfer efficiency of 77.8% (14/18) and 76.1% (10/13), respectively. The isolates from the environment conjugated with a significant more efficiency than those from the GIT [Two-sample test of proportions; p-value = 0.0119]. The overall conjugation transfer frequencies ranged from 0.4 × 10-14 - 5.5 × 10-11 per donor cells with the highest median conjugation transfer frequency observed among isolates from animal (3.23 × 10-12 [IQR: 0.70 × 10-12 - 7.22 × 10-12]) followed by that of isolates from the environment (1.60 × 10-12 [IQR: 0.30 × 10-12 - 5.0 × 10-12]). ESBL producing E. coli from human, animals and environment exercises horizontal blaCTX-M gene transfer efficiently with the highest occurrence among isolates from the environment and animals. The antimicrobial resistance control and prevention strategies should be widened up to explore strategies to prevent horizontal AMR gene transfer.

Effect of resistance difference on distribution of antibiotics in bacterial cell and conjugative gene transfer risks during electrochemical flow through reaction

The occurrences and spread of antibiotic resistance (AR) mediated by horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs) in aquatic environment have been aggravated because of the abuse of antibiotics. While the pressure of different antibiotics is known to induce the spread of AR in bacteria, whether distribution of different antibiotics in cell structure could affect HGT risks is not clear. Here, a significant difference between the distribution of tetracycline hydrochloride (Tet) and sulfamethoxazole (Sul) in cell structure during electrochemical flow through reaction (EFTR) process was firstly reported. Meanwhile, EFTR treatment possessed excellent disinfection performance and consequently controlled the HGT risks. The intracellular Tet (iTet) was discharged through efflux pumps to increase the content of extracellular Tet (eTet) due to the resistance of donor E. coli DH5α under the selective pressure of Tet, declining the damage of donor and plasmid RP4. The HGT frequency was 8.18-fold increase compared with that by EFTR treatment alone. While the secretion of intracellular Sul (iSul) was inhibited by blocking the formation of efflux pumps to inactivate the donor under the Sul pressure, and the total content of iSul and adsorbed Sul (aSul) to be 1.36-fold higher than that of eSul. Therefore, the reactive oxygen species (ROS) generation and cell membrane permeability were improved to release ARGs, and •OH attacked plasmid RP4 in the EFTR process, inhibiting the HGT risks. This study advances the awareness of the interaction between distribution of different antibiotics in cell structure and the HGT risks in the EFTR process.

The DNA Phosphorothioation Restriction-Modification System Influences the Antimicrobial Resistance of Pathogenic Bacteria.

Bacterial defense barriers, such as DNA methylation-associated restriction-modification (R-M) and the CRISPR-Cas system, play an important role in bacterial antimicrobial resistance (AMR). Recently, a novel R-M system based on DNA phosphorothioate (PT) modification has been shown to be widespread in the kingdom of Bacteria as well as Archaea. However, the potential role of the PT R-M system in bacterial AMR remains unclear. In this study, we explored the role of PT R-Ms in AMR with a series of common clinical pathogenic bacteria. By analyzing the distribution of AMR genes related to mobile genetic elements (MGEs), it was shown that the presence of PT R-M effectively reduced the distribution of horizontal gene transfer (HGT)-derived AMR genes in the genome, even in the bacteria that did not tend to acquire AMR genes by HGT. In addition, unique gene variation analysis based on pangenome analysis and MGE prediction revealed that the presence of PT R-M could suppress HGT frequency. Thus, this is the first report showing that the PT R-M system has the potential to repress HGT-derived AMR gene acquisition by reducing the HGT frequency. IMPORTANCE In this study, we demonstrated the effect of DNA PT modification-based R-M systems on horizontal gene transfer of AMR genes in pathogenic bacteria. We show that there is no apparent association between the genetic background of the strains harboring PT R-Ms and the number of AMR genes or the kinds of gene families. The strains equipped with PT R-M harbor fewer plasmid-derived, prophage-derived, or integrating mobile genetic element (iMGE)-related AMR genes and have a lower HGT frequency, but the degree of inhibition varies among different bacteria. In addition, compared with Salmonella enterica and Escherichia coli, Klebsiella pneumoniae prefers to acquire MGE-derived AMR genes, and there is no coevolution between PT R-M clusters and bacterial core genes.

Evaluating the occurrence frequency of horizontal gene transfer induced by different degrees of heavy metal stress

Horizontal gene transfer of heavy metal resistance genes and antibiotic resistance genes in bacteria frequently occurred during composting. However, the relevancy between heavy metal pollution with different degrees and the occurrence of horizontal gene transfer during composting was unclear. Therefore, this study aimed to clarify the underlying relationship. The changes of resistance gene abundance and network analysis results showed that heavy metal pollution induced high level of resistance gene abundance in microorganisms and promoted the occurrence of horizontal gene transfer. Furthermore, regression fitting models indicated that the relevancy between resistance genes abundance and horizontal gene transfer under high pollution (HP: R = 0.90) was weakener than that under low pollution (LP: R = 0.95) and medium pollution (MP: R = 0.97). Ultimately, structural equation models further verified that the abundance of resistance genes was mainly caused by horizontal gene transfer during LP (coefficients: 0.997) and MP (coefficients: 0.993) composting. However, the high level of resistance gene abundance was caused by host bacteria (coefficients: 0.775) and horizontal gene transfer (coefficients: 0.701) during HP composting. The serious heavy metal pollution had selection pressure on host bacteria to induce them to develop resistance during HP composting, rather than only through horizontal gene transfer. Therefore, mild heavy metal pollution was more liable to cause horizontal gene transfer and increase the spread risk of resistance genes during composting. This study helped to better understand the risks caused by heavy metal pollution by evaluating the frequency of horizontal gene transfer at different pollution levels during composting.

A Novel Module Promotes Horizontal Gene Transfer in Azorhizobium caulinodans ORS571.

Azorhizobium caulinodans ORS571 contains an 87.6 kb integrative and conjugative element (ICEAc) that conjugatively transfers symbiosis genes to other rhizobia. Many hypothetical redundant gene fragments (rgfs) are abundant in ICEAc, but their potential function in horizontal gene transfer (HGT) is unknown. Molecular biological methods were employed to delete hypothetical rgfs, expecting to acquire a minimal ICEAc and consider non-functional rgfs as editable regions for inserting genes related to new symbiotic functions. We determined the significance of rgf4 in HGT and identified the physiological function of genes designated rihF1a (AZC_3879), rihF1b (AZC_RS26200), and rihR (AZC_3881). In-frame deletion and complementation assays revealed that rihF1a and rihF1b work as a unit (rihF1) that positively affects HGT frequency. The EMSA assay and lacZ-based reporter system showed that the XRE-family protein RihR is not a regulator of rihF1 but promotes the expression of the integrase (intC) that has been reported to be upregulated by the LysR-family protein, AhaR, through sensing host’s flavonoid. Overall, a conservative module containing rihF1 and rihR was characterized, eliminating the size of ICEAc by 18.5%. We propose the feasibility of constructing a minimal ICEAc element to facilitate the exchange of new genetic components essential for symbiosis or other metabolic functions between soil bacteria.

Massive annotation of bacterial l-asparaginases reveals their puzzling distribution and frequent gene transfer events

l-Asparaginases, which convert l-asparagine to l-aspartate and ammonia, come in five types, AI-AV. Some bacterial type AII enzymes are a key element in the treatment of acute lymphoblastic leukemia in children, but new l-asparaginases with better therapeutic properties are urgently needed. Here, we search publicly available bacterial genomes to annotate l-asparaginase proteins belonging to the five known types. We characterize taxonomic, phylogenetic, and genomic patterns of l-asparaginase occurrences pointing to frequent horizontal gene transfer (HGT) events, also occurring multiple times in the same recipient species. We show that the reference AV gene, encoding a protein originally found and structurally studied in Rhizobium etli, was acquired via HGT from Burkholderia. We also describe the sequence variability of the five l-asparaginase types and map the conservation levels on the experimental or predicted structures of the reference enzymes, finding the most conserved residues in the protein core near the active site, and the most variable ones on the protein surface. Additionally, we highlight the most common sequence features of bacterial AII proteins that may aid in selecting therapeutic l-asparaginases. Finally, we point to taxonomic units of bacteria that do not contain recognizable sequences of any of the known l-asparaginase types, implying that those microorganisms most likely contain new, as yet unknown types of l-asparaginases. Such novel enzymes, when properly identified and characterized, could hold promise as antileukemic drugs.

Loss of the Rhodobacter capsulatus Serine Acetyl Transferase Gene, cysE1, Impairs Gene Transfer by Gene Transfer Agents and Biofilm Phenotypes.

ABSTRACTBiofilms are widespread in the environment, where they allow bacterial species to survive adverse conditions. Cells in biofilms are densely packed, and this proximity is likely to increase the frequency of horizontal gene transfer. Gene transfer agents (GTAs) are domesticated viruses with the potential to spread any gene between bacteria. GTA production is normally restricted to a small subpopulation of bacteria, and regulation of GTA loci is highly coordinated, but the environmental conditions that favor GTA production are poorly understood. Here, we identified a serine acetyltransferase gene, cysE1, in Rhodobacter capsulatus that is required for optimal receipt of GTA DNA, accumulation of extracellular polysaccharide, and biofilm formation. The cysE1 gene is directly downstream of the core Rhodobacter-like GTA (RcGTA) structural gene cluster and upregulated in an RcGTA overproducer strain, although it is expressed on a separate transcript. The data we present suggest that GTA production and biofilm are coregulated, which could have important implications for the study of rapid bacterial evolution and understanding the full impact of GTAs in the environment.IMPORTANCE Direct exchange of genes between bacteria leads to rapid evolution and is the major factor underlying the spread of antibiotic resistance. Gene transfer agents (GTAs) are an unusual but understudied mechanism for genetic exchange that are capable of transferring any gene from one bacterium to another, and therefore, GTAs are likely to be important factors in genome plasticity in the environment. Despite the potential impact of GTAs, our knowledge of their regulation is incomplete. In this paper, we present evidence that elements of the cysteine biosynthesis pathway are involved in coregulation of various phenotypes required for optimal biofilm formation by Rhodobacter capsulatus and successful infection by the archetypal RcGTA. Establishing the regulatory mechanisms controlling GTA-mediated gene transfer is a key stepping stone to allow a full understanding of their role in the environment and wider impact.

Prevalence and mobility of integrative and conjugative elements within a Streptomyces natural population.

Horizontal Gene Transfer (HGT) is a powerful force generating genomic diversity in bacterial populations. HGT in Streptomyces is in large part driven by conjugation thanks to plasmids, Integrative and Conjugative elements (ICEs) and Actinomycete ICEs (AICEs). To investigate the impact of ICE and AICE conjugation on Streptomyces genome evolution, we used in silico and experimental approaches on a set of 11 very closely related strains isolated from a millimeter scale rhizosphere population. Through bioinformatic searches of canonical conjugation proteins, we showed that AICEs are the most frequent integrative conjugative elements, with the central chromosome region being a hotspot for integrative element insertion. Strains exhibited great variation in AICE composition consistent with frequent HGT and/or gene loss. We found that single insertion sites can be home to different elements in different strains (accretion) and conversely, elements belonging to the same family can be found at different insertion sites. A wide variety of cargo genes was present in the AICEs with the potential to mediate strain-specific adaptation (e.g., DNA metabolism and resistance genes to antibiotic and phages). However, a large proportion of AICE cargo genes showed hallmarks of pseudogenization, consistent with deleterious effects of cargo genes on fitness. Pock assays enabled the direct visualization of conjugal AICE transfer and demonstrated the transfer of AICEs between some, but not all, of the isolates. Multiple AICEs were shown to be able to transfer during a single mating event. Although we did not obtain experimental evidence for transfer of the sole chromosomal ICE in this population, genotoxic stress mediated its excision from the chromosome, suggesting its functionality. Our results indicate that AICE-mediated HGT in Streptomyces populations is highly dynamic, with likely impact on strain fitness and the ability to adapt to environmental change.