A preliminary study of α-amidoamids as new potential antimicrobial drugs was performed. Special emphasis was placed on selection of structure of α-amidoamids with the highest biological activity against different types of Gram-stained bacteria by lipopolysaccharide (LPS). Herein, Escherichia coli model strains K12 (without LPS in its structure) and R1-R4 (with different length LPS in its structure) were used. The presented work showed that the antibacterial activity of α-amidoamids depends on their structure and affects the LPS of bacteria. Moreover, the influence of various newly synthesized α-amidoamids on bacteria possessing smooth and rought LPS and oxidative damage of plasmid DNA caused by all newly obtained compounds was indicated. The presented studies clearly explain that α-amidoamids can be used as substitutes for antibiotics. The chemical and biological activity of the analysed α-amidoamids was associated with short alkyl chain and different isocyanides molecules in their structure such as: tetr-butyl isocyanide or 2,5-dimethoxybenzyl isocyanide. The observed results are especially important in the case of the increasing resistance of bacteria to various drugs and antibiotics.

New methodology must be developed to improve the ability to characterize the growing number of amino acid sequences, which vastly exceeds the number of experimentally determined protein structures. Homologous proteins can be used as structural templates for modeling proteins that do not have experimentally determined structures. However, in many cases, there are no homologous proteins (typically <30% sequence identity) with determined structures from which a query sequence can be reliably modeled. The aim of protein threading is to use features, such as secondary structure, solvent accessibility and torsional angles, in addition to sequence patterns to identify structural templates from the protein databank to assist for full-length atomic-level structural modeling. However, there are still numerous protein sequences for which correct templates cannot be recognized. This raises the question as to what attributes allow query sequences to be matched to the correct but distantly homologous templates. To aid the investigation into this question and to provide genome-score protein structure for the biological community, a database called THE-DB (threading hard and easy protein database) has been developed in which it becomes possible to analyze over 15 000 query sequences from the Escherichia coli (E. coli) K12 and human proteomes, as well as to find their three-dimensional templates derived from the state-of-the-art threading algorithms which is not feasible with existing protein template databases. The E. coli K12 and human data can be downloaded in bulk from the THE-DB page.

Lactones are among the well-known organic substances with a specific taste and smell. They are characterized by antibacterial, antiviral, anti-inflammatory, and anti-cancer properties. In recent years, among this group of compounds, new biologically active substances have been searched by modifying the main (leading) structure with new analogs with stronger or different responses that may have a toxic effect on the cells of pathogenic bacteria and constitute an alternative to commonly used antibiotics. A preliminary study of δ-lactone derivatives as new potential candidates for antibacterial drugs was conducted. Particular emphasis was placed on the selection of the structure of lactones with the highest biological activity, especially those with fluorine in their structure as a substituent in terms of action on bacterial lipopolysaccharide (LPS) in the model strains of Escherichia coli K12 (without LPS in its structure) and R2-R4 (LPS of different lengths in its structure). In the presented studies, on the basis of the conducted MIC and MBC tests, it was shown that the antibacterial (toxic) activity of lactones depends on their structure and the length of the bacterial LPS in the membrane of specific strains. Moreover, oxidative damage of bacterial DNA isolated from bacteria after modification with newly synthesized compounds after application of the repair enzyme Fpg glycosylase was analyzed. The analyzed damage values were compared with the modification with appropriate antibiotics: ciprofloxacin, bleomycin, and cloxacillin. The presented research clearly shows that lactone derivatives can be potential candidates as substitutes for drugs, e.g., the analyzed antibiotics. Their chemical and biological activity is related to coumarin derivatives and the corresponding δ-lactone groups in the structure of the substituent. The observed results are of particular importance in the case of increasing bacterial resistance to various drugs and antibiotics, especially in nosocomial infections and neoplasms, and in the era of a microbial pandemic.

The number of small proteins (SPs) encoded in the Escherichia coli genome is unknown, as current bioinformatics and biochemical techniques make short gene and small protein identification challenging. One method of small protein identification involves adding an epitope tag to the 3' end of a short open reading frame (sORF) on the chromosome, with synthesis confirmed by immunoblot assays. In this study, this strategy was used to identify new E. coli small proteins, tagging 80 sORFs in the E. coli genome, and assayed for protein synthesis. The selected sORFs represent diverse sequence characteristics, including degrees of sORF conservation, predicted transmembrane domains, sORF direction with respect to flanking genes, ribosome binding site (RBS) prediction, and ribosome profiling results. Of 80 sORFs, 36 resulted in encoded synthesized proteins-a 45% success rate. Modeling of detected versus non-detected small proteins analysis showed predictions based on RBS prediction, transcription data, and ribosome profiling had statistically-significant correlation with protein synthesis; however, there was no correlation between current sORF annotation and protein synthesis. These results suggest substantial numbers of small proteins remain undiscovered in E. coli, and existing bioinformatics techniques must continue to improve to facilitate identification.

Diarrhea caused by pathogenic Escherichia coli (E. coli) is one of the most serious infectious diseases in humans and animals. Due to antibiotics resistance and the lack of efficient vaccine, more attention should be paid to find potential versatile vaccine candidates to prevent diseases. In this study, the sequence homology analysis indicated that OmpF from E. coli CVCC 1515 shares a high identity (90-100%) with about half of the E. coli (46.7%) and Shigella (52.8%) strains. Then the recombinant OmpF was supposed to be developed as a versatile vaccine to prevent E. coli infection. OmpF was expressed in E. coli BL21 (DE3) using the auto-induction method. The recombinant OmpF (rOmpF) protein had an average molecular weight of 40 kDa with the purity of 90%. Immunological analysis indicated that the titers of anti-rOmpF sera against rOmpF and whole cells were 1:240,000 and 1:27,000, respectively. The opsonophagocytosis result showed that 72.21 ± 11.39 and 11.04 ± 3.90% of bacteria were killed in the rOmpF immunization and control groups, respectively. The survival ratio of mice immunized with rOmpF ranged between 40 and 60% as observed within 36 h after challenge, indicating mice were partially protected from E. coli CVCC 1515 infection. The expressed rOmpF protein induced an effective immune response, but only provide a weak protection against pathogenic E. coli CVCC 1515 and a small reduction in E. coli CICC 21530 (O157:H7) excretion in a mouse infection model. Native forms of the OmpF antigen may be studied for immunogenicity and potential protective efficacy.

In modern times, bacterial infections have become a growing problem in the medical community due to the emergence of antibiotic-resistant bacteria. In fact, the overuse and improper disposal of antibiotics have led to bacterial resistance and the presence of such bacteria in wastewater. Therefore, it is critical to develop effective strategies for dealing with antibiotic-resistant bacteria in wastewater. Electroporation has been found to be one of the most promising complementary techniques for bacterial inactivation because it is effective against a wide range of bacteria, is non-chemical and is highly optimizable. Many studies have demonstrated electroporation-assisted inactivation of bacteria, but rarely have clinical antibiotics or bacteria resistant to these antibiotics been used in the study. Therefore, the motivation for our study was to use a treatment regimen that combines antibiotics and electroporation to inactivate antibiotic-resistant bacteria.

sInterBase is a comprehensive and easy-to-operate web-based platform for mining experimentally identified sRNA-mRNA interactions in Escherichia coli. Interactions in the database are annotated with an interaction duplex and a set of descriptive features. sInterBase provides advanced functionality, such as flexible search based on various criteria, statistical analysis via charts, browsing, and downloading interactions for further use.

Bacterial resistance has become a serious threat to human health. In particular, the gradual development of resistance to polymyxins, the last line of defense for human infections, is a major issue. Secreted proteins contribute to the interactions between bacteria and the environment. In this study, we compared the secretomes of polymyxin B-sensitive and -resistant Escherichia coli strains by data-independent acquisition mass spectrometry. In total, 87 differentially expressed secreted proteins were identified in polymyxin B-resistant E. coli compared to the sensitive strain. A GO enrichment analysis indicated that the differentially expressed proteins were involved in biological processes, including bacterial-type flagellum-dependent cell motility, ion transport, carbohydrate derivative biosynthetic process, cellular response to stimulus, organelle organization, and cell wall organization or biogenesis. The differentially expressed secreted proteins in polymyxin B-resistant bacteria were enriched for multiple pathways, suggesting that the resistance phenotype depends on complex regulatory mechanisms. A potential biomarker or drug target (YebV) was found in polymyxin B-resistant E. coli. This work clarifies the secretome changes associated with the acquisition of polymyxin resistance and may contribute to drug development.

Escherichia coli strain LS5218 is a useful host for the production of fatty acid derived products, but the genetics underlying this utility have not been fully investigated. Here, we report the genome sequence of LS5218 and a list of large mutations and single nucleotide permutations (SNPs) relative to E. coli K-12 strain MG1655. We discuss how genetic differences may affect the physiological differences between LS5218 and MG1655. We find that LS5218 is more closely related to E. coli strain NCM3722 and suspect that small genetic differences between K-12 derived strains may have a significant impact on metabolic engineering efforts.

Conflicts between replication and transcription challenge chromosome duplication. Escherichia coli replisome movement along transcribed DNA is promoted by Rep and UvrD accessory helicases with Δrep ΔuvrD cells being inviable under rapid growth conditions. We have discovered that mutations in a tRNA gene, aspT, in an aminoacyl tRNA synthetase, AspRS, and in a translation factor needed for efficient proline-proline bond formation, EF-P, suppress Δrep ΔuvrD lethality. Thus replication-transcription conflicts can be alleviated by the partial sacrifice of a mechanism that reduces replicative barriers, namely translating ribosomes that reduce RNA polymerase backtracking. Suppression depends on RelA-directed synthesis of (p)ppGpp, a signalling molecule that reduces replication-transcription conflicts, with RelA activation requiring ribosomal pausing. Levels of (p)ppGpp in these suppressors also correlate inversely with the need for Rho activity, an RNA translocase that can bind to emerging transcripts and displace transcription complexes. These data illustrate the fine balance between different mechanisms in facilitating gene expression and genome duplication and demonstrate that accessory helicases are a major determinant of this balance. This balance is also critical for other aspects of bacterial survival: the mutations identified here increase persistence indicating that similar mutations could arise in naturally occurring bacterial populations facing antibiotic challenge.

The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles.

A previously reported multi-scale model for (ultra-)small-angle X-ray (USAXS/SAXS) and (very) small-angle neutron scattering (VSANS/SANS) of live Escherichia coli was revised on the basis of compositional/metabolomic and ultrastructural constraints. The cellular body is modeled, as previously described, by an ellipsoid with multiple shells. However, scattering originating from flagella was replaced by a term accounting for the oligosaccharide cores of the lipopolysaccharide leaflet of the outer membrane including its cross-term with the cellular body. This was mainly motivated by (U)SAXS experiments showing indistinguishable scattering for bacteria in the presence and absence of flagella or fimbrae. The revised model succeeded in fitting USAXS/SAXS and differently contrasted VSANS/SANS data of E. coli ATCC 25922 over four orders of magnitude in length scale. Specifically, this approach provides detailed insight into structural features of the cellular envelope, including the distance of the inner and outer membranes, as well as the scattering length densities of all bacterial compartments. The model was also successfully applied to E. coli K12, used for the authors' original modeling, as well as for two other E. coli strains. Significant differences were detected between the different strains in terms of bacterial size, intermembrane distance and its positional fluctuations. These findings corroborate the general applicability of the approach outlined here to quantitatively study the effect of bactericidal compounds on ultrastructural features of Gram-negative bacteria without the need to resort to any invasive staining or labeling agents.

Escherichia coli K1 is the leading cause of meningitis in newborns. Understanding the molecular basis of E. coli K1 pathogenicity will help develop treatment of meningitis and prevent neurological sequelae. E. coli K1 replicates in host blood and forms a high level of bacteremia to cause meningitis in human. However, the mechanisms that E. coli K1 employs to sense niche signals for survival in host blood are poorly understood. We identified one intergenic region in E. coli K1 genome that encodes a novel small RNA, sRNA-17. The expression of sRNA-17 was downregulated by ArcA in microaerophilic blood. The ΔsRNA-17 strain grew better in blood than did the wild-type strain and enhanced invasion frequency in human brain microvascular endothelial cells. Transcriptome analyses revealed that sRNA-17 regulates tens of differentially expressed genes. These data indicate that ArcA downregulates the sRNA-17 expression to benefit bacterial survival in blood and penetration of the blood-brain barrier. Our findings reveal a signaling mechanism in E. coli K1 for host adaptation.

The structure of dynamic folds in microbial chromosomes is largely unknown. Here, we find that genes with a highly biased codon composition and characterizing a functional core in Escherichia coli K12 show to be periodically distributed along the arcs, suggesting an encoded three-dimensional genomic organization helping functional activities among which are translation and, possibly, transcription. This extends to functional classes of genes that are shown to systematically organize into two independent positional gene networks, one driven by metabolic genes and the other by genes involved in cellular processing and signaling. We conclude that functional reasons justify periodic gene organization. This finding generates new questions on evolutionary pressures imposed on the chromosome. Our methodological approach is based on single genome analysis. Given either core genes or genes organized in functional classes, we analyze the detailed distribution of distances between pairs of genes through a parameterized model based on signal processing and find that these groups of genes tend to be separated by a regular integral distance. The methodology can be applied to any set of genes and can be taken as a footprint for large-scale bacterial and archaeal analysis.

Although genes on the chromosome are organized in a fixed order, the spatial correlations in transcription have not been systematically evaluated. We used a combination of genomic and signal processing techniques to investigate the properties of transcription in the genome of Escherichia coli K12 as a function of the position of genes on the chromosome.

The achievement of an active biological entity from environmental DNA is important in the field of phage. In this study, the environmental DNA extracted from hospital wastewater was transferred into Escherichia coli DH10B and Escherichia coli BL21 with chemical transformation and electroporation. After transformation, overnight cultures were filtered and used as phage source. The efficacies of the techniques were evaluated with spot test and double-layer agar assay. The emerged phage, named as ADUt, was purified and host-range analysis was performed. Phage DNA was isolated, sequenced and restriction profile was determined. The genome was assembled. The phylogenetic tree was constructed via VipTree. The extracted DNA resulted in active phage by the transformation of E. coli DH10B, but not E. coli BL21. The chemical transformation was found more successful than electroporation. ADUt phage was found to be polyvalent and effective against limited strains of Shigella and Escherichia genera. The phage genome size and GC ratio are 166904 bp and 35.67%, respectively. ADUt is a member of Straboviridae family and Tequatrovirus genus. This is the first study that uses environmental DNA for acquiring active phage, which may be an important source of new phage discovery. The result showed that DNA transformation yields active bacteriophage with both chemical transformation and electroporation.

Enteroaggregative Escherichia coli (EAEC) are important diarrhoeal pathogens that are defined by a HEp-2 adherence assay performed in specialist laboratories. Multilocus sequence typing (MLST) has revealed that aggregative adherence is convergent, providing an explanation for why not all EAEC hybridize with the plasmid-derived probe for this category, designated CVD432. Some EAEC lineages are globally disseminated or more closely associated with disease.

Thymol is a phenolic compound used for its wide spectrum antimicrobial activity. There is a limited understanding of the antimicrobial mechanisms underlying thymol activity. To investigate this, E. coli strain JM109 was exposed to thymol at sub-lethal concentrations and after 16 rounds of exposure, isolates with a 2-fold increased minimal inhibitory concentration (MIC) were recovered (JM109-Thyr). The phenotype was stable after multiple sub-cultures without thymol.

Horizontally acquired genes are usually transcriptionally inactive, although most of them are associated with genomic loci enriched with promoter-like sequences forming "promoter islands." We hypothesized that lateral DNA transfer induces local mutagenesis, accumulating AT base pairs and creating promoter-like sequences, whose occupancy with RNA polymerase and a specific silencer H-NS suppresses the transcription of foreign genes. Error-prone mutagenesis was implemented for the "promoter island" of a foreign gene appY and the promoter region of an inherent gene dps. Derivatives with changed transcriptional activity were selected using a reporter plasmid pET28_eGFP. Only one cycle of mutagenesis with negative selection suppressed the activity of the main dps promoter to the background level due to a single substitution in its -10 element, while positive selection gave a sequence with improved -35 element, thus testifying feasibility of the approach. The same suppression for appY was achieved by three cycles, while eightfold transcription activation required nine iterations of mutagenesis. In both cases, the number of potential start points decreased resulting in an ordinary regulatory region with only one dominant promoter in the case of positive selection. Efficiency of H-NS binding remained virtually unchanged in all mutant constructs. Based on these findings we conclude that excessive promoters can adversely affect transcription by providing a platform for interference between several RNA polymerase molecules, which can act as a silencer at promoter-dense regions.

In Escherichia coli, approximately 100 regulatory small RNAs (sRNAs) have been identified experimentally and many more have been predicted by various methods. To provide a comprehensive overview of sRNAs, we analysed the low-molecular-weight RNAs (< 200 nt) of E. coli with deep sequencing, because the regulatory RNAs in bacteria are usually 50-200 nt in length.