RAPP-containing arrest peptides induce translational stalling by short circuiting the ribosomal peptidyltransferase activity.

Arrest peptides containing RAPP (ArgAlaProPro) motifs have been discovered in both Gram-positive and Gram-negative bacteria, where they are thought to regulate expression of important protein localization machinery components. Here we determine cryo-EM structures of ribosomes stalled on RAPP arrest motifs in both Bacillus subtilis and Escherichia coli. Together with molecular dynamics simulations, our structures reveal that the RAPP motifs allow full accommodation of the A-site tRNA, but prevent the subsequent peptide bond from forming. Our data support a model where the RAP in the P-site interacts and stabilizes a single hydrogen atom on the Pro-tRNA in the A-site, thereby preventing an optimal geometry for the nucleophilic attack required for peptide bond formation to occur. This mechanism to short circuit the ribosomal peptidyltransferase activity is likely to operate for the majority of other RAPP-like arrest peptides found across diverse bacterial phylogenies.

Alternate routes to mnm5s2U synthesis in Gram-positive bacteria.

The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNAGlu, Gln, Asp contains a variety of xnm5s2U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm5s2U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm5s2U to mnm5s2U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm5s2U in both Bacillus subtilis and Streptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm5s2U into mnm5s2U in B. subtilis. Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature.IMPORTANCEThe xnm5s2U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm5s2U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

Clostridium lamae sp. nov., a novel bacterium isolated from the fresh feces of alpaca.

A novel Gram-positive, anaerobic, nonspore-forming, rod-shaped bacterium, designated strain NGMCC 1.200840 T, was isolated from the alpacas fresh feces. The taxonomic position of the novel strain was determined using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed strain NGMCC 1.200840 T was a member of the genus Clostridium and closely related to Clostridium tertium DSM 2485 T (98.16% sequence similarity). Between strains NGMCC 1.200840 T and C. tertium DSM 2485 T, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were 79.91% and 23.50%, respectively. Genomic DNA G + C content is 28.44 mol%. The strain can utilise D-glucose, D-mannitol, D-lactose, D-saccharose, D-maltose, D-xylose, L-arabinose, D-cellobiose, D-mannose, D-melezitose, D-raffinose, D-sorbitol, L-rhamnose, D-trehalose, D-galactose and Arbutin to produce acid. The optimal growth pH was 7, the temperature was 37 °C, and the salt concentration was 0-0.5% (w/v). The major cellular fatty acids (> 10%) included iso-C15:0, anteiso-C15:0 and iso-C17:0 3-OH. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unidentified phospholipids and two unidentified aminolipids. Based on phenotypic, phylogenetic and chemotaxonomic characteristics, NGMCC 1.200840 T represents a novel species within the genus Clostridium, for which the named Clostridium lamae sp. nov. is proposed. The type strain is NGMCC 1.200840 T (= CGMCC 1.18014 T = JCM 35704 T).

Genetic Engineering and Rebooting of Bacteriophages in L-Form Bacteria.

The rapid increase of circulating, antibiotic-resistant pathogens is a major ongoing global health crisis, and arguably, the end of the "golden age of antibiotics" is looming. This has led to a surge in research and development of alternative antimicrobials, including bacteriophages, to treat such infections (phage therapy). Isolating natural phage variants for the treatment of individual patients is an arduous and time-consuming task. Furthermore, the use of natural phages is frequently hampered by natural limitations, such as moderate in vivo activity, the rapid emergence of resistance, insufficient host range, or the presence of undesirable genetic elements within the phage genome. Targeted genetic editing of wild-type phages (phage engineering) has successfully been employed in the past to mitigate some of these pitfalls and to increase the therapeutic efficacy of the underlying phage variants. Clearly, there is a large potential for the development of novel, marker-less genome-editing methodologies to facilitate the engineering of therapeutic phages. Steady advances in synthetic biology have facilitated the in vitro assembly of modified phage genomes, which can be activated ("rebooted") upon transformation of a suitable host cell. However, this can prove challenging, especially in difficult-to-transform Gram-positive bacteria. In this chapter, we detail the production of cell wall-deficient L-form bacteria and their application to activate synthetic genomes of phages infecting Gram-positive host species.

[Mechanism of high-frequent horizontal gene transfer in Gram positive bacterial pathogens].

Horizontal gene transfer through transconjugation and natural transformation plays a major role in the spread of antimicrobial resistance. Although the phenomenon of genetic element transmission has long been known, the rapid increase in the number of antimicrobial resistant bacteria in recent years and the accompanying accumulation of genomic information have revealed that horizontal gene transfer contributes to genome plasticity in various ways. The author reported the molecular mechanism of the antimicrobial activity of the accessory factor bacteriocin encoded by the junctional transfer plasmid of Enterococcus faecalis, a representative Gram-positive opportunistic pathogen that is concerned as highly antimicrobial resistant, and found diversity in the selfimmune system based on epidemiological studies. In addition, the author established a technique to visualize and quantify genomic recombination by natural transformation in Streptococcus pneumoniae which is also one of the most concerns for antimicrobial resistance and vaccine escape, at single cells level resolution in real time. Focuses on outcome from these research, this paper introduces the molecular mechanisms that promote horizontal gene transmission and the prospects for their technological application.

Models of Hfq interactions with small non-coding RNA in Gram-negative and Gram-positive bacteria.

Hfq is required by many Gram-negative bacteria to chaperone the interaction between small non-coding RNA (sRNA) and mRNA to facilitate annealing. Conversely and despite the presence of Hfq in many Gram-positive bacteria, sRNAs in Gram-positive bacteria bind the mRNA target independent of Hfq. Details provided by the Hfq structures from both Gram-negative and Gram-positive bacteria have demonstrated that despite a conserved global structure of the protein, variations of residues on the binding surfaces of Hfq results in the recognition of different RNA sequences as well as the ability of Hfq to facilitate the annealing of the sRNA to the mRNA target. Additionally, a subset of Gram-negative bacteria has an extended C-terminal Domain (CTD) that has been shown to affect the stability of the Hfq hexamer and increase the rate of release of the annealed sRNA-mRNA product. Here we review the structures of Hfq and biochemical data that have defined the interactions of the Gram-negative and Gram-positive homologues to highlight the similarities and differences in the interactions with RNA. These interactions provided a deeper understanding of the how Hfq functions to facilitate the annealing of sRNA-mRNA, the selectivity of the interactions with RNA, and the role of the CTD of Hfq in the interactions with sRNA.

Genome mining reveals secondary metabolites of Antarctic bacterium Streptomyces albidoflavus related to antimicrobial and antiproliferative activities.

The urgent need for new antimicrobials arises from antimicrobial resistance. Actinobacteria, especially Streptomyces genus, are responsible for production of numerous clinical antibiotics and anticancer agents. Genome mining reveals the biosynthetic gene clusters (BGCs) related to secondary metabolites and the genetic potential of a strain to produce natural products. However, this potential may not be expressed under laboratory conditions. In the present study, the Antarctic bacterium was taxonomically affiliated as Streptomyces albidoflavus ANT_B131 (CBMAI 1855). The crude extracts showed antimicrobial activity against both fungi, Gram-positive and Gram-negative bacteria and antiproliferative activity against five human tumor cell lines. Whole-genome sequencing reveals a genome size of 6.96 Mb, and the genome mining identified 24 BGCs, representing 13.3% of the genome. The use of three culture media and three extraction methods reveals the expression and recovery of 20.8% of the BGCs. The natural products identified included compounds, such as surugamide A, surugamide D, desferrioxamine B + Al, desferrioxamine E, and ectoine. This study reveals the potential of S. albidoflavus ANT_B131 as a natural product producer. Yet, the diversity of culture media and extraction methods could enhance the BGCs expression and recovery of natural products, and could be a strategy to intensify the BGC expression of natural products.

Structural basis for the selective methylation of 5-carboxymethoxyuridine in tRNA modification.

Posttranscriptional modifications of tRNA are widely conserved in all domains of life. Especially, those occurring within the anticodon often modulate translational efficiency. Derivatives of 5-hydroxyuridine are specifically found in bacterial tRNA, where 5-methoxyuridine and 5-carboxymethoxyuridine are the major species in Gram-positive and Gram-negative bacteria, respectively. In certain tRNA species, 5-carboxymethoxyuridine can be further methylated by CmoM to form the methyl ester. In this report, we present the X-ray crystal structure of Escherichia coli CmoM complexed with tRNASer1, which contains 5-carboxymethoxyuridine at the 5'-end of anticodon (the 34th position of tRNA). The 2.22 Å resolution structure of the enzyme-tRNA complex reveals that both the protein and tRNA undergo local conformational changes around the binding interface. Especially, the hypomodified uracil base is flipped out from the canonical stacked conformation enabling the specific molecular interactions with the enzyme. Moreover, the structure illustrates that the enzyme senses exclusively the anticodon arm region of the substrate tRNA and examines the presence of key determinants, 5-carboxymethoxyuridine at position 34 and guanosine at position 35, offering molecular basis for the discriminatory mechanism against non-cognate tRNAs.

Comparative evaluation of MALDI-TOF MS and 16S rRNA gene sequencing for the identification of clinically relevant anaerobic bacteria: critical evaluation of discrepant results.

OBJECTIVES: The main study objective was to evaluate the correlation between matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing results for the identification of anaerobes. METHODS: A retrospective study was conducted of all anaerobic bacteria isolated from clinically significant specimens. MALDI-TOF (Bruker Byotyper) and 16S rRNA gene sequencing were performed in all strains. Identifications were considered correct when the concordance with gene sequencing was ≥99%. RESULTS: The study included 364 isolates of anaerobic bacteria: 201 (55.2%) Gram-negative and 163 (44.8%) Gram-positive, mostly belonging to the genus Bacteroides. Isolates were largely obtained from blood cultures (128/35.4%) and intra-abdominal samples (116/32.1%). Overall, 87.3% of isolates were identified at species level using the version 9 database (89.5% of Gram-negative and 84.6% of Gram-positive anaerobic bacteria). All isolates belonging to the species B. fragilis sensu stricto were correctly identified by MALDI-TOF MS, but five cases of Phocaeicola (Bacteroides) dorei were misidentified as Phocaeicola (Bacteroides) vulgatus; all Prevotella isolates were correctly identified at the genus level, and most were correctly identified at the species level. Among Gram-positive anaerobes, 12 Anaerococcus species were not identified by MALDI-TOF MS, while six cases identified as Peptoniphilus indolicus were found to belong to other genera/species. CONCLUSIONS: MALDI-TOF is a reliable technique for identifying most anaerobic bacteria, although the database needs frequent updating to identify rare, infrequent, and newly discovered species.

Role of bacterial efflux pump proteins in antibiotic resistance across microbial species.

Efflux proteins are transporter molecules that actively pump out a variety of substrates, including antibiotics, from cells to the environment. They are found in both Gram-positive and Gram-negative bacteria and eukaryotic cells. Based on their protein sequence homology, energy source, and overall structure, efflux proteins can be divided into seven groups. Multidrug efflux pumps are transmembrane proteins produced by microbes to enhance their survival in harsh environments and contribute to antibiotic resistance. These pumps are present in all bacterial genomes studied, indicating their ancestral origins. Many bacterial genes encoding efflux pumps are involved in transport, a significant contributor to antibiotic resistance in microbes. Efflux pumps are widely implicated in the extrusion of clinically relevant antibiotics from cells to the extracellular environment and, as such, represent a significant challenge to antimicrobial therapy. This review aims to provide an overview of the structures and mechanisms of action, substrate profiles, regulation, and possible inhibition of clinically relevant efflux pumps. Additionally, recent advances in research and the pharmacological exploitation of efflux pump inhibitors as a promising intervention for combating drug resistance will be discussed.

Development of a Novel Phagomagnetic-Assisted Isothermal DNA Amplification System for Endpoint Electrochemical Detection of Listeria monocytogenes.

The hitherto implemented Listeria monocytogenes detection techniques are cumbersome or require expensive non-portable instrumentation, hindering their transposition into on-time surveillance systems. The current work proposes a novel integrated system resorting to loop-mediated isothermal amplification (LAMP), assisted by a bacteriophage P100-magnetic platform, coupled to an endpoint electrochemical technique, towards L. monocytogenes expeditious detection. Molybdophosphate-based optimization of the bacterial phagomagnetic separation protocol allowed the determination of the optimal parameters for its execution (pH 7, 25 °C, 32 µg of magnetic particles; 60.6% of specific capture efficiency). The novel LAMP method targeting prfA was highly specific, accomplishing 100% inclusivity (for 61 L. monocytogenes strains) and 100% exclusivity (towards 42 non-target Gram-positive and Gram-negative bacteria). As a proof-of-concept, the developed scheme was successfully validated in pasteurized milk spiked with L. monocytogenes. The phagomagnetic-based approach succeeded in the selective bacterial capture and ensuing lysis, triggering Listeria DNA leakage, which was efficiently LAMP amplified. Methylene blue-based electrochemical detection of LAMP amplicons was accomplished in 20 min with remarkable analytical sensitivity (1 CFU mL-1). Hence, the combined system presented an outstanding performance and robustness, providing a 2.5 h-swift, portable, cost-efficient detection scheme for decentralized on-field application.

Total Bacterial RNA Isolation and Northern Blotting Analysis.

The study of bacterial gene expression during infection provides vital information for researchers to understand bacterial pathogenesis and infection. The ability to obtain clean and undegraded RNA could be challenging and daunting and remains the most crucial experimental step prior to downstream analyses, such as Northern blotting, quantitative PCR (qPCR), and RNA-seq.This chapter describe two methods (acid guanidinium thiocyanate (TRIzol) phenol-chloroform and hot phenol) commonly used to isolate total bacterial RNA and are suitable for both Gram-positive and Gram-negative bacteria. Procedures such as RNA quantification and DNase treatment are also included to ensure amount and quality of the RNA samples. The second part of the chapter includes a method used to analyze bacterial gene expression (Northern blotting), two methods to generate radioactive probes, as well as target detection using a phosphorimager.

A survey on antimicrobial resistance genes of frequently used probiotic bacteria, 1901 to 2022.

BackgroundAntimicrobial resistance (AMR) is caused by AMR determinants, mainly genes (ARGs) in the bacterial genome. Bacteriophages, integrative mobile genetic elements (iMGEs) or plasmids can allow ARGs to be exchanged among bacteria by horizontal gene transfer (HGT). Bacteria, including bacteria with ARGs, can be found in food. Thus, it is conceivable that in the gastrointestinal tract, bacteria from the gut flora could take up ARGs from food.AimThe study objective was to gain insight into the ARG set carried by commonly used probiotic bacteria that may enter the human body with non-fermented foods, fermented foods, or probiotic dietary supplements (FFPs) and to assess ARG mobility.MethodsNext generation sequencing whole genome data from 579 isolates of 12 commonly employed probiotic bacterial species were collected from a public repository. Using bioinformatical tools, ARGs were analysed and linkage with mobile genetic elements assessed.ResultsResistance genes were found in eight bacterial species. The ratios of ARG positive/negative samples per species were: Bifidobacterium animalis (65/0), Lactiplantibacillus plantarum (18/194), Lactobacillus delbrueckii (1/40), Lactobacillus helveticus (2/64), Lactococcus lactis (74/5), Leucoconstoc mesenteroides (4/8), Levilactobacillus brevis (1/46), Streptococcus thermophilus (4/19). In 66% (112/169) of the ARG-positive samples, at least one ARG could be linked to plasmids or iMGEs. No bacteriophage-linked ARGs were found.ConclusionThe finding of potentially mobile ARGs in probiotic strains for human consumption raises awareness of a possibility of ARG HGT in the gastrointestinal tract. In addition to existing recommendations, screening FFP bacterial strains for ARG content and mobility characteristics might be considered.

Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR.

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.

Multi-drug resistant ESKAPE pathogens and the uses of plants as their antimicrobial agents.

Infections by ESKAPE (Enterococcus sp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens cause major concern due to their multi-drug resistance (MDR). The ESKAPE pathogens are frequently linked to greater mortality, diseases, and economic burden in healthcare worldwide. Therefore, the use of plants as a natural source of antimicrobial agents provide a solution as they are easily available and safe to use. These natural drugs can also be enhanced by incorporating silver nanoparticles and combining them with existing antibiotics. By focussing the attention on the ESKAPE organisms, the MDR issue can be addressed much better.

Streptomyces isolated from bird feathers as a potential source for novel antibiotics.

Streptomyces, the main source of antibiotics essential for human health, are widely distributed in nature among terrestrial, oceanic and atmospheric environments. New trends in antibiotic discovery are focused in the search for novel bioactive strains in unexplored habitats. We provide here evidence of the presence of diverse Streptomyces populations in wild bird feathers, such as the seagull, Larus michahellis, collected at Northern Spain; the sparrow, Passer domesticus, and the hoopoe, Upupa epops, both collected in Southern Spain. Taxonomic identification of fourteen bioactive strains, by sequencing their 16S rRNA gene and phylogenetic analyses, revealed that all of them are homologous to a total of 10 different Streptomyces. Strains from seagull samples are homologous to other antibiotic producers previously isolated from atmospheric, marine and terrestrial environments in the Cantabrian Sea region, Northern Spain. Isolates form Southern feather samples, from a house sparrow and a Eurasian hoopoe, are homologues to Streptomyces strains previously isolated mainly from soils along the Mediterranean region. The most relevant feature is that they are producers of diverse antibiotics with activity against Gram-positive, Gram-negative bacteria and fungi. We report here the successful activation of silent antibiotic biosynthetic pathways in response to changes in environmental conditions, such as incubation temperature and salinity of the culture medium, in agreement with the OSMAC approach, One Strain Many Compounds. The finding of bioactive Streptomyces in bird's plumage might be of relevance, not only in the ecology of Streptomyces-birds associations, but also in medicine and biotechnology since they can be regarded as a potential source for novel antibiotics.

A widespread family of phage-inducible chromosomal islands only steals bacteriophage tails to spread in nature.

Phage satellites are genetic elements that couple their life cycle to that of helper phages they parasitize, interfering with phage packaging through the production of small capsids, where only satellites are packaged. So far, in all analyzed systems, the satellite-sized capsids are composed of phage proteins. Here, we report that a family of phage-inducible chromosomal islands (PICIs), a type of satellites, encodes all the proteins required for both the production of small-sized capsids and the exclusive packaging of the PICIs into these capsids. Therefore, this new family, named capsid-forming PICIs (cf-PICIs), only requires phage tails to generate PICI particles. Remarkably, the representative cf-PICIs are produced with no cost from their helper phages, suggesting that the relationship between these elements is not parasitic. Finally, our phylogenomic studies indicate that cf-PICIs are present both in gram-positive and gram-negative bacteria and have evolved at least three times independently to spread in nature.

Two broadly conserved families of polyprenyl-phosphate transporters.

Peptidoglycan and almost all surface glycopolymers in bacteria are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP)1-4. These UndP-linked precursors are transported across the membrane and polymerized or directly transferred to surface polymers, lipids or proteins. UndP is then flipped to regenerate the pool of cytoplasmic-facing UndP. The identity of the flippase that catalyses transport has remained unknown. Here, using the antibiotic amphomycin that targets UndP5-7, we identified two broadly conserved protein families that affect UndP recycling. One (UptA) is a member of the DedA superfamily8; the other (PopT) contains the domain DUF368. Genetic, cytological and syntenic analyses indicate that these proteins are UndP transporters. Notably, homologues from Gram-positive and Gram-negative bacteria promote UndP transport in Bacillus subtilis, indicating that recycling activity is broadly conserved among family members. Inhibitors of these flippases could potentiate the activity of antibiotics targeting the cell envelope.

Isolation and Characterization of Bacteria Associated with Onion and First Report of Onion Diseases Caused by Five Bacterial Pathogens in Texas, U.S.A.

Bacterial diseases pose a severe challenge to growers and cause significant loss to the billion-dollar onion industry in the United States. Texas is the sixth largest onion producing state, yet the bacterial communities associated with short-day onion crops grown in Texas have not been studied. This study was conducted to identify, characterize, and understand the diversity of bacteria associated with onion production in Texas. In 2020, 190 foliar and 210 bulb samples were collected from onion crops in the Rio Grande Valley and Winter Garden regions of Texas. Sequencing of the 16s rRNA gene was used to identify each bacterial strains to a genus. The pathogenicity to onion of each bacterial strain was tested using three assays: a red onion scale assay, a yellow onion bulb assay, and a foliar assay. Whole genome sequencing was done to identify the onion-pathogenic strains to species. Collectively, isolates of 24 genera belonging to three phyla were detected, including 19 genera from foliar samples and nine genera from bulb samples. Isolates in the Phylum Proteobacteria, including 15 genera of Gram-negative bacteria, were the most abundant of the taxa, comprising 90.0% of the strains isolated. The diversity of foliar isolates was evenly distributed between Gram-positive and Gram-negative bacteria, while Gram-negative bacteria dominated the isolates from bulb samples. In total, 83.9% of the bacterial isolates were not pathogenic on onion, with only isolates of Pantoea, Pseudomonas, Burkholderia, Erwinia, Enterobacter, and Curtobacterium proving pathogenic. Strains of Burkholderia gladioli, Pseudomonas alliivorans, Pantoea agglomerans, P. ananatis, and P. allii are the first documented cases of these pathogens of onion in Texas. Identifying and characterizing the nature of onion microflora, including pathogens of onion, is vital to developing rapid disease detection techniques via pathogenomics and minimizing losses through the application of effective disease management measures.

Structure-function studies reveal ComEA contains an oligomerization domain essential for transformation in gram-positive bacteria.

An essential step in bacterial transformation is the uptake of DNA into the periplasm, across the thick peptidoglycan cell wall of Gram-positive bacteria, or the outer membrane and thin peptidoglycan layer of Gram-negative bacteria. ComEA, a DNA-binding protein widely conserved in transformable bacteria, is required for this uptake step. Here we determine X-ray crystal structures of ComEA from two Gram-positive species, Bacillus subtilis and Geobacillus stearothermophilus, identifying a domain that is absent in Gram-negative bacteria. X-ray crystallographic, genetic, and analytical ultracentrifugation (AUC) analyses reveal that this domain drives ComEA oligomerization, which we show is required for transformation. We use multi-wavelength AUC (MW-AUC) to characterize the interaction between DNA and the ComEA DNA-binding domain. Finally, we present a model for the interaction of the ComEA DNA-binding domain with DNA, suggesting that ComEA oligomerization may provide a pulling force that drives DNA uptake across the thick cell walls of Gram-positive bacteria.