Low-Toxicity Self-Photosensitized Biohybrid Systems for Enhanced Light-Driven H2 Production.

Nanoparticles (NPs) represent a potential optoelectronic source capable of significantly boosting hydrogen production; however, their inevitable cytotoxicity may lead to oxidative damage of bacterial cell membranes. In this study, we employed non-photosynthetic Escherichia coli K-12 as a model organism and utilized self-assembled cadmium sulfide (CdS) nanoparticles to construct a low-toxicity and hydrogen-production-enhancing self-photosensitive hybrid system. To mitigate the cytotoxicity of CdS NPs and synthesize biocompatible CdS NPs on the cell surface, we employed engineered E. coli (efeB/OE) for bioremediation, achieving this goal through the overexpression of the peroxidase enzyme (EfeB). A comparative analysis with E. coli-CdS revealed a significant downregulation of genes encoding oxidative stress proteins in efeB/OE-CdS post-irradiation. Atomic force microscopy (AFM) confirmed the stability of bacterial cell membranes. Due to the enhanced stability of the cell membrane, the hydrogen yield of the efeB/OE-CdS system increased by 1.3 times compared to the control, accompanied by a 49.1% reduction in malondialdehyde (MDA) content. This study proposes an effective strategy to alleviate the toxicity of mixed biological nanoparticle systems and efficiently harness optoelectronic electrons, thereby achieving higher hydrogen production in bioremediation.

One-pot synthesis of alginate-antimicrobial peptide nanogel.

Nanosized alginate-based particles (NAPs) were obtained in a one-pot solvent-free synthesis procedure, achieving the design of a biocompatible nanocarrier for the encapsulation of IbM6 antimicrobial peptide (IbM6). IbM6 is integrated in the nascent nanosized hydrogel self-assembly guided by electrostatic interactions and by weak interactions, typical of soft matter. The formation of the nanogel is a dynamic and complex process, which presents an interesting temporal evolution. In this work, we optimized the synthesis conditions of IbM6-NAPs based on small-angle X-ray scattering (SAXS) measurements and evaluated its time evolution over several weeks by sensing the IbM6 environment in IbM6-NAPs from photochemical experiments. Fluorescence deactivation experiments revealed that the accessibility of different quenchers to the IbM6 peptide embedded in NAPs is dependent on the aging time of the alginate network. Lifetimes measurements indicate that the deactivation paths of the excited state of the IbM6 in the nanoaggregates are reduced when compared with those exhibited by the peptide in aqueous solution, and are also dependent on the aging time of the nanosized alginate network. Finally, the entrapment of IbM6 in NAPs hinders the degradation of the peptide by trypsin, increasing its antimicrobial activity against Escherichia coli K-12 in simulated operation conditions.

Discovery of a Gut Bacterial Metabolic Pathway that Drives α-Synuclein Aggregation.

Parkinson's disease (PD) etiology is associated with aggregation and accumulation of α-synuclein (α-syn) proteins in midbrain dopaminergic neurons. Emerging evidence suggests that in certain subtypes of PD, α-syn aggregates originate in the gut and subsequently spread to the brain. However, mechanisms that instigate α-syn aggregation in the gut have remained elusive. In the brain, the aggregation of α-syn is induced by oxidized dopamine. Such a mechanism has not been explored in the context of the gastrointestinal tract, a niche harboring 46% of the body's dopamine reservoirs. Here, we report that Enterobacteriaceae, a bacterial family prevalent in human gut microbiotas, induce α-syn aggregation. More specifically, our in vitro data indicate that respiration of nitrate by Escherichia coli K-12, which results in production of nitrite that mediates oxidation of Fe2+ to Fe3+, creates an oxidizing redox potential. These oxidizing conditions enabled the formation of dopamine-derived quinones and α-syn aggregates. Exposing nitrite, but not nitrate, to enteroendocrine STC-1 cells induced aggregation of α-syn that is natively expressed in these cells, which line the intestinal tract. Taken together, our findings indicate that bacterial nitrate reduction may be critical for initiating intestinal α-syn aggregation.

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.

The intrinsically disordered linker in the single-stranded DNA-binding protein influences DNA replication restart and recombination pathways in Escherichia coli K-12.

Tetrameric single-stranded (ss) DNA-binding proteins (SSBs) stabilize ssDNA intermediates formed during genome maintenance reactions in Bacteria. SSBs also recruit proteins important for these processes through direct SSB-protein interactions, including proteins involved in DNA replication restart and recombination processes. SSBs are composed of an N-terminal oligomerization and ssDNA-binding domain, a C-terminal acidic tip that mediates SSB-protein interactions, and an internal intrinsically disordered linker (IDL). Deletions and insertions into the IDL are well tolerated with few phenotypes, although the largest deletions and insertions exhibit some sensitivity to DNA-damaging agents. To define specific DNA metabolism processes dependent on IDL length, ssb mutants that lack 16, 26, 37, or 47 residues of the 57-residue IDL were tested for synthetic phenotypes with mutations in DNA replication restart or recombination genes. We also tested the impact of integrating a fluorescent domain within the SSB IDL using an ssb::mTur2 insertion mutation. Only the largest deletion tested or the insertion mutation causes sensitivity in any of the pathways. Mutations in two replication restart pathways (PriA-B1 and PriA-C) showed synthetic lethalities or small colony phenotypes with the largest deletion or insertion mutations. Recombination gene mutations del(recBCD) and del(ruvABC) show synthetic phenotypes only when combined with the largest ssb deletion. These results suggest that a minimum IDL length is important in some genome maintenance reactions in Escherichia coli. These include pathways involving PriA-PriB1, PriA-PriC, RecFOR, and RecG. The mTur2 insertion in the IDL may also affect SSB interactions in some processes, particularly the PriA-PriB1 and PriA-PriC replication restart pathways.IMPORTANCEssb is essential in Escherichia coli due to its roles in protecting ssDNA and coordinating genome maintenance events. While the DNA-binding core and acidic tip have well-characterized functions, the purpose of the intrinsically disordered linker (IDL) is poorly understood. In vitro studies have revealed that the IDL is important for cooperative ssDNA binding and phase separation. However, single-stranded (ss) DNA-binding protein (SSB) variants with large deletions and insertions in the IDL support normal cell growth. We find that the PriA-PriB1 and PriA-C replication restart, as well as the RecFOR- and RecG-dependent recombination, pathways are sensitive to IDL length. This suggests that cooperativity, phase separation, or a longer spacer between the core and acidic tip of SSB may be important for specific cellular functions.

Diversity, Distribution, and Chromosomal Rearrangements of TRIP1 Repeat Sequences in Escherichia coli.

The bacterial genome contains numerous repeated sequences that greatly affect its genomic plasticity. The Escherichia coli K-12 genome contains three copies of the TRIP1 repeat sequence (TRIP1a, TRIP1b, and TRIP1c). However, the diversity, distribution, and role of the TRIP1 repeat sequence in the E. coli genome are still unclear. In this study, after screening 6725 E. coli genomes, the TRIP1 repeat was found in the majority of E. coli strains (96%: 6454/6725). The copy number and direction of the TRIP1 repeat sequence varied in each genome. Overall, 2449 genomes (36%: 2449/6725) had three copies of TRIP1 (TRIP1a, TRIP1b, and TRIP1c), which is the same as E. coli K-12. Five types of TRIP1 repeats, including two new types (TRIP1d and TRIP1e), are identified in E. coli genomes, located in 4703, 3529, 5741, 1565, and 232 genomes, respectively. Each type of TRIP1 repeat is localized to a specific locus on the chromosome. TRIP1 repeats can cause intra-chromosomal rearrangements. A total of 156 rearrangement events were identified, of which 88% (137/156) were between TRIP1a and TRIP1c. These findings have important implications for future research on TRIP1 repeats.

Pre-clinical safety assessment of biotechnologically produced lacto-N-tetraose (LNT).

Lacto-N-tetraose (LNT) is a human milk oligosaccharide with average concentrations ranging from 0.74 to 1.07 g/L in breastmilk, depending on the lactation stage. In this study, the preclinical safety of LNT produced by the Escherichia coli K-12 E2083 production strain was assessed. LNT was negative in both the bacterial reverse mutation assay and the in vitro micronucleus assay, demonstrating the absence of genotoxic potential for this substance. In the OECD 408 guideline compliant 90-day oral toxicity study rat, LNT did not induce any adverse effects in any treatment group up to and including the highest dose tested, and no LOAEL could be determined. Therefore, the no-observed-adverse effect level (NOAEL) is set at the highest dose level tested, i.e. a dietary level of 5 % (w/w), corresponding to ≥2856 mg/kg bw/day and ≥3253 mg/kg bw/day for males and females, respectively. This might be an underestimation of the NOAEL, caused by the range of dose levels tested. The results obtained in the current study are in good agreement with available data generated using other biotechnologically produced LNT batches and therefore support its safe use as a food ingredient.

Analyzing the correlation between protein expression and sequence-related features of mRNA and protein in Escherichia coli K-12 MG1655 model.

It was necessary to have a tool that could predict the amount of protein and optimize the gene sequences to produce recombinant proteins efficiently. The Transim model published by Tuller et al. in 2018 can calculate the translation rate in E. coli using features on the mRNA sequence, achieving a Spearman correlation with the amount of protein per mRNA of 0.36 when tested on the dataset of operons' first genes in E. coli K-12 MG1655 genome. However, this Spearman correlation was not high, and the model did not fully consider the features of mRNA and protein sequences. Therefore, to enhance the prediction capability, our study firstly tried expanding the testing dataset, adding genes inside the operon, and using the microarray of the mRNA expression data set, thereby helping to improve the correlation of translation rate with the amount of protein with more than 0.42. Next, the applicability of 6 traditional machine learning models to calculate a "new translation rate" was examined using initiation rate and elongation rate as inputs. The result showed that the SVR algorithm had the most correlated new translation rates, with Spearman correlation improving to R = 0.6699 with protein level output and to R = 0.6536 with protein level per mRNA. Finally, the study investigated the degree of improvement when combining more features with the new translation rates. The results showed that the model's predictive ability to produce a protein per mRNA reached R = 0.6660 when using six features, while the correlation of this model's final translation rate to protein level was up to R = 0.6729. This demonstrated the model's capability to predict protein expression of a gene, rather than being limited to predicting expression by an mRNA and showed the model's potential for development into gene expression predicting tools.

Unlocking the potential of Escherichia coli K-12: A novel approach for malignancy reduction in colorectal cancer through gene expression modulation.

Studies have noted the association between Escherichia coli K-12 (E. coli K-12) and the reduction of malignancy in colorectal cancer (CRC). However, the molecular mechanisms underlying this relationship have not been thoroughly explored. The aim of this study was to identify the genes influenced by E. coli K-12 and their connection to CRC. We identified the genes affected by E. coli K-12 using the GSE50040 dataset. Additionally, we investigated the relationship between the expression of genes affected by E. coli K-12 and CRC using the cancer genome atlas data. The association between the expression of E. coli K-12-affected genes and patient prognosis was investigated using clinical data. Pathways related to CRC and E. coli K-12-related genes were analyzed using the Enrichr tool. Furthermore, we employed a protein-protein interaction (PPI) network to identify hub genes associated with both E. coli K-12 and CRC. To validate our findings, we conducted RT-qPCR analysis on CRC samples and adjacent normal tissue. The results of GSE50040 showed that E. coli K-12 could change the expression of many genes related to CRC in colorectal cell lines. The results showed that E. coli K-12 reduces the expression of several genes linked to the main pathways used by cancer cells, such as the metastasis, WNT, cell proliferation pathway, and mTORC1. It was demonstrated that elevated BGN, FJX1, and LZTS1 expression is linked to a bad prognosis in patients and that E. coli K-12 may be able to lower this expression. Also, based on the PPI network, genes such as KLF4 and CXCL3 were identified as hub genes related to genes affected by E. coli K-12. When KLF4 and CXCL3 expression levels in cancer samples were compared to nearby normal tissue, a significant change in these genes' expression levels was found in CRC. Our findings demonstrated the potential relationship between oncogene genes and genes impacted by E. coli K-12. Also, our findings demonstrated that E. coli K-12 may regulate the expression of genes linked to a high death rate. In summary, the results of this study suggest that E. coli K-12 can be regarded as a significant probiotic with the potential to mitigate the risk of CRC development.

Proteomics-based vaccine targets annotation and design of multi-epitope vaccine against antibiotic-resistant Streptococcus gallolyticus.

Streptococcus gallolyticus is a non-motile, gram-positive bacterium that causes infective endocarditis. S. gallolyticus has developed resistance to existing antibiotics, and no vaccine is currently available. Therefore, it is essential to develop an effective S. gallolyticus vaccine. Core proteomics was used in this study together with subtractive proteomics and reverse vaccinology approach to find antigenic proteins that could be utilized for the design of the S. gallolyticus multi-epitope vaccine. The pipeline identified two antigenic proteins as potential vaccine targets: penicillin-binding protein and the ATP synthase subunit. T and B cell epitopes from the specific proteins were forecasted employing several immunoinformatics and bioinformatics resources. A vaccine (360 amino acids) was created using a combination of seven cytotoxic T cell lymphocyte (CTL), three helper T cell lymphocyte (HTL), and five linear B cell lymphocyte (LBL) epitopes. To increase immune responses, the vaccine was paired with a cholera enterotoxin subunit B (CTB) adjuvant. The developed vaccine was highly antigenic, non-allergenic, and stable for human use. The vaccine's binding affinity and molecular interactions with the human immunological receptor TLR4 were studied using molecular mechanics/generalized Born surface area (MMGBSA), molecular docking, and molecular dynamic (MD) simulation analyses. Escherichia coli (strain K12) plasmid vector pET-28a ( +) was used to examine the ability of the vaccine to be expressed. According to the outcomes of these computer experiments, the vaccine is quite promising in terms of developing a protective immunity against diseases. However, in vitro and animal research are required to validate our findings.

Immunoinformatics-guided approach for designing a pan-proteome multi-epitope subunit vaccine against African swine fever virus.

Despite being identified over a hundred years ago, there is still no commercially available vaccine for the highly contagious and deadly African swine fever virus (ASFV). This study used immunoinformatics for the rapid and inexpensive designing of a safe and effective multi-epitope subunit vaccine for ASFV. A total of 18,858 proteins from 100 well-annotated ASFV proteomes were screened using various computational tools to identify potential epitopes, or peptides capable of triggering an immune response in swine. Proteins from genotypes I and II were prioritized for their involvement in the recent global ASFV outbreaks. The screened epitopes exhibited promising qualities that positioned them as effective components of the ASFV vaccine. They demonstrated antigenicity, immunogenicity, and cytokine-inducing properties indicating their ability to induce potent immune responses. They have strong binding affinities to multiple swine allele receptors suggesting a high likelihood of yielding more amplified responses. Moreover, they were non-allergenic and non-toxic, a crucial prerequisite for ensuring safety and minimizing any potential adverse effects when the vaccine is processed within the host. Integrated with an immunogenic 50S ribosomal protein adjuvant and linkers, the epitopes formed a 364-amino acid multi-epitope subunit vaccine. The ASFV vaccine construct exhibited notable immunogenicity in immune simulation and molecular docking analyses, and stable profiles in secondary and tertiary structure assessments. Moreover, this study designed an optimized codon for efficient translation of the ASFV vaccine construct into the Escherichia coli K-12 expression system using the pET28a(+) vector. Overall, both sequence and structural evaluations suggested the potential of the ASFV vaccine construct as a candidate for controlling and eradicating outbreaks caused by the pathogen.

Fitness trade-offs of multidrug efflux pumps in Escherichia coli K-12 in acid or base, and with aromatic phytochemicals.

Multidrug efflux pumps are the frontline defense mechanisms of Gram-negative bacteria, yet little is known of their relative fitness trade-offs under gut conditions such as low pH and the presence of antimicrobial food molecules. Low pH contributes to the proton-motive force (PMF) that drives most efflux pumps. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. Competition assays were performed by flow cytometry of co-cultured Escherichia coli K-12 strains possessing or lacking a given pump complex. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with carbonyl cyanide 3-chlorophenylhydrazone or at pH 8.0). At pH 5.5, selection against AcrAB-TolC was increased by aromatic acids, alcohols, and related phytochemicals such as methyl salicylate. The degree of fitness cost for AcrA was correlated with the phytochemical's lipophilicity (logP). Methyl salicylate and salicylamide selected strongly against AcrA, without genetic induction of drug resistance regulons. MdtEF-TolC and EmrAB-TolC each had a fitness cost at pH 5.5, but salicylate or benzoate made the fitness contribution positive. Pump fitness effects were not explained by gene expression (measured by digital PCR). Between pH 5.5 and 8.0, acrA and emrA were upregulated in the log phase, whereas mdtE expression was upregulated in the transition-to-stationary phase and at pH 5.5 in the log phase. Methyl salicylate did not affect pump gene expression. Our results suggest that lipophilic non-acidic molecules select against a major efflux pump without inducing antibiotic resistance regulons.IMPORTANCEFor drugs that are administered orally, we need to understand how ingested phytochemicals modulate drug resistance in our gut microbiome. Bacteria maintain low-level resistance by proton-motive force (PMF)-driven pumps that efflux many different antibiotics and cell waste products. These pumps play a key role in bacterial defense by conferring resistance to antimicrobial agents at first exposure while providing time for a pathogen to evolve resistance to higher levels of the antibiotic exposed. Nevertheless, efflux pumps confer energetic costs due to gene expression and pump energy expense. The bacterial PMF includes the transmembrane pH difference (ΔpH), which may be depleted by permeant acids and membrane disruptors. Understanding the fitness costs of efflux pumps may enable us to develop resistance breakers, that is, molecules that work together with antibiotics to potentiate their effect. Non-acidic aromatic molecules have the advantage that they avoid the Mar-dependent induction of regulons conferring other forms of drug resistance. We show that different pumps have distinct selection criteria, and we identified non-acidic aromatic molecules as promising candidates for drug resistance breakers.

RegulonDB v12.0: a comprehensive resource of transcriptional regulation in E. coli K-12.

RegulonDB is a database that contains the most comprehensive corpus of knowledge of the regulation of transcription initiation of Escherichia coli K-12, including data from both classical molecular biology and high-throughput methodologies. Here, we describe biological advances since our last NAR paper of 2019. We explain the changes to satisfy FAIR requirements. We also present a full reconstruction of the RegulonDB computational infrastructure, which has significantly improved data storage, retrieval and accessibility and thus supports a more intuitive and user-friendly experience. The integration of graphical tools provides clear visual representations of genetic regulation data, facilitating data interpretation and knowledge integration. RegulonDB version 12.0 can be accessed at https://regulondb.ccg.unam.mx.

Functional annotation of enzyme-encoding genes using deep learning with transformer layers.

Functional annotation of open reading frames in microbial genomes remains substantially incomplete. Enzymes constitute the most prevalent functional gene class in microbial genomes and can be described by their specific catalytic functions using the Enzyme Commission (EC) number. Consequently, the ability to predict EC numbers could substantially reduce the number of un-annotated genes. Here we present a deep learning model, DeepECtransformer, which utilizes transformer layers as a neural network architecture to predict EC numbers. Using the extensively studied Escherichia coli K-12 MG1655 genome, DeepECtransformer predicted EC numbers for 464 un-annotated genes. We experimentally validated the enzymatic activities predicted for three proteins (YgfF, YciO, and YjdM). Further examination of the neural network's reasoning process revealed that the trained neural network relies on functional motifs of enzymes to predict EC numbers. Thus, DeepECtransformer is a method that facilitates the functional annotation of uncharacterized genes.

Engineering the synthetic ß-alanine pathway in Komagataella phaffii for conversion of methanol into 3-hydroxypropionic acid.

BACKGROUND: Methanol is increasingly gaining attraction as renewable carbon source to produce specialty and commodity chemicals, as it can be generated from renewable sources such as carbon dioxide (CO2). In this context, native methylotrophs such as the yeast Komagataella phaffii (syn Pichia pastoris) are potentially attractive cell factories to produce a wide range of products from this highly reduced substrate. However, studies addressing the potential of this yeast to produce bulk chemicals from methanol are still scarce. 3-Hydroxypropionic acid (3-HP) is a platform chemical which can be converted into acrylic acid and other commodity chemicals and biopolymers. 3-HP can be naturally produced by several bacteria through different metabolic pathways. RESULTS: In this study, production of 3-HP via the synthetic ß-alanine pathway has been established in K. phaffii for the first time by expressing three heterologous genes, namely panD from Tribolium castaneum, yhxA from Bacillus cereus, and ydfG from Escherichia coli K-12. The expression of these key enzymes allowed a production of 1.0 g l-1 of 3-HP in small-scale cultivations using methanol as substrate. The addition of a second copy of the panD gene and selection of a weak promoter to drive expression of the ydfG gene in the PpCß21 strain resulted in an additional increase in the final 3-HP titer (1.2 g l-1). The 3-HP-producing strains were further tested in fed-batch cultures. The best strain (PpCß21) achieved a final 3-HP concentration of 21.4 g l-1 after 39 h of methanol feeding, a product yield of 0.15 g g-1, and a volumetric productivity of 0.48 g l-1 h-1. Further engineering of this strain aiming at increasing NADPH availability led to a 16% increase in the methanol consumption rate and 10% higher specific productivity compared to the reference strain PpCß21. CONCLUSIONS: Our results show the potential of K. phaffii as platform cell factory to produce organic acids such as 3-HP from renewable one-carbon feedstocks, achieving the highest volumetric productivities reported so far for a 3-HP production process through the ß-alanine pathway.

utPCR: A Strategy for the Highly Specific and Absolutely Quantitative Detection of Single Molecules within Only Minutes.

Bloodstream infection is a major health problem worldwide, with extremely high mortality. Detecting infection in the early stage is challenging due to the extremely low concentration of bacteria in the blood. Digital PCR provides unparalleled sensitivity and can achieve absolute quantification, but it is time-consuming. Moreover, the presence of unavoidable background signals in negative controls poses a significant challenge for single-molecule detection. Here, we propose a novel strategy called "Ultrafast flexible thin tube-based droplet digital PCR (utPCR)" that can shorten the digital PCR process from 2 h to only 5 min, with primer annealing/extension time reduced from minutes to only 5 s. Importantly, the ultrafast PCR eliminates nonspecific amplification and thus enables single-molecule detection. The utPCR enabled the sensitive detection and digital quantification of E. coli O157 in the high background of a 106-fold excess of E. coli K12 cells. Moreover, this method also displayed the potential to detect rare pathogens in blood samples, and the limit of detection (LOD) could be as low as 10 CFU per mL of blood without false positive results. Considered ultrafast (<5 min) and highly sensitive (single-molecule detection), the utPCR holds excellent prospects in the next generation of molecular diagnosis.

O antigen biogenesis sensitises Escherichia coli K-12 to bile salts, providing a plausible explanation for its evolutionary loss.

Escherichia coli K-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However, Escherichia coli K-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies have reported the presence of independent mutations that abolish OAg repeating-unit (RU) biogenesis in E. coli K-12 strains from the same original source, suggesting unknown evolutionary forces have selected for inactivation of OAg biogenesis during the early propagation of K-12. Here, we show for the first time that restoration of OAg in E. coli K-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS primarily contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-linked OAg intermediates in the periplasmic leaflet of the inner membrane interfere with peptidoglycan sacculus biosynthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for as an evolutionary fitness benefit. Our work thus provides a plausible explanation for the long-held mystery of the selective pressure that may have led to the loss of OAg biogenesis in E. coli K-12; this opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria.

The timing of transcription of RpoS-dependent genes varies across multiple stresses in Escherichia coli K-12.

IMPORTANCE: Bacteria adapt to changing environments by altering the transcription of their genes. Specific proteins can regulate these changes. This study explored how a single protein called RpoS controls how many genes change expression during adaptation to three stresses. We found that: (i) RpoS is responsible for activating different genes in different stresses; (ii) that during a stress, the timing of gene activation depends on the what stress it is; and (iii) that how much RpoS a gene needs in order to be activated can predict when that gene will be activated during the stress of stationary phase.

Bacteria metabolic adaptation to oxidative stress: the case of silica.

Although the presence of silica in many living organisms offers advanced properties including cell protection, the different in vitro attempts to build living materials in pure silica never favoured the cells viability. Thus, little attention has been paid to host-guest interactions to modify the expected biologic response. Here we report the physiological changes undergone by Escherichia coli K-12 in silica from colloidal solution to gel confinement. We show that the physiological alterations in growing cultures are not triggered by the initial oxidative Reactive Oxygen Species (ROS) response. Silica promotes the induction of alternative metabolic pathways along with an increase of growth suggesting the existence of rpoS polymorphisms. Since the functionality of hybrid materials depends on the specific biologic responses of their guests, such cell physiological adaptation opens perspectives in the design of bioactive devices attracting for a large field of sciences.

Mechanisms of Regulation of Cryptic Prophage-Encoded Gene Products in Escherichia coli.

The dicBF operon of Qin cryptic prophage in Escherichia coli K-12 encodes the small RNA (sRNA) DicF and small protein DicB, which regulate host cell division and are toxic when overexpressed. While new functions of DicB and DicF have been identified in recent years, the mechanisms controlling the expression of the dicBF operon have remained unclear. Transcription from dicBp, the major promoter of the dicBF operon, is repressed by DicA. In this study, we discovered that transcription of the dicBF operon and processing of the polycistronic mRNA is regulated by multiple mechanisms. DicF sRNA accumulates during stationary phase and is processed from the polycistronic dicBF mRNA by the action of both RNase III and RNase E. DicA-mediated transcriptional repression of dicBp can be relieved by an antirepressor protein, Rem, encoded on the Qin prophage. Ectopic production of Rem results in cell filamentation due to strong induction of the dicBF operon, and filamentation is mediated by DicF and DicB. Spontaneous derepression of dicBp occurs in a subpopulation of cells independent of the antirepressor. This phenomenon is reminiscent of the bistable switch of λ phage with DicA and DicC performing functions similar to those of CI and Cro, respectively. Additional experiments demonstrate stress-dependent induction of the dicBF operon. Collectively, our results illustrate that toxic genes carried on cryptic prophages are subject to layered mechanisms of control, some that are derived from the ancestral phage and some that are likely later adaptations. IMPORTANCE Cryptic or defective prophages have lost genes necessary to excise from the bacterial chromosome and produce phage progeny. In recent years, studies have found that cryptic prophage gene products influence diverse aspects of bacterial host cell physiology. However, to obtain a complete understanding of the relationship between cryptic prophages and the host bacterium, identification of the environmental, host, or prophage-encoded factors that induce the expression of cryptic prophage genes is crucial. In this study, we examined the regulation of a cryptic prophage operon in Escherichia coli encoding a small RNA and a small protein that are involved in inhibiting bacterial cell division, altering host metabolism, and protecting the host bacterium from phage infections.