Elucidation of the Gemcitabine Transporters of Escherichia coli K-12 and Gamma-Proteobacteria Linked to Gemcitabine-Related Chemoresistance.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine), a widely used anticancer drug, is considered a gold standard in treating aggressive pancreatic cancers. Gamma-proteobacteria that colonize the pancreatic tumors contribute to chemoresistance against gemcitabine by metabolizing the drug to a less active and deaminated form. The gemcitabine transporters of these bacteria are unknown to date. Furthermore, there is no complete knowledge of the gemcitabine transporters in Escherichia coli or any other related proteobacteria. In this study, we investigate the complement of gemcitabine transporters in E. coli K-12 and two common chemoresistance-related bacteria (Klebsiella pneumoniae and Citrobacter freundii). We found that E. coli K-12 has two high-affinity gemcitabine transporters with distinct specificity properties, namely, NupC and NupG, whereas the gemcitabine transporters of C. freundii and K. pneumoniae include the NupC and NupG orthologs, functionally indistinguishable from their counterparts, and, in K. pneumoniae, one additional NupC variant, designated KpNupC2. All these bacterial transporters have a higher affinity for gemcitabine than their human counterparts. The highest affinity (KM 2.5-3.0 µΜ) is exhibited by NupGs of the bacteria-specific nucleoside-H+ symporter (NHS) family followed by NupCs (KM 10-13 µΜ) of the concentrative nucleoside transporter (CNT) family, 15-100 times higher than the affinities reported for the human gemcitabine transporter hENT1/SLC29A1, which is primarily associated with gemcitabine uptake in the pancreatic adenocarcinoma cells. Our results offer a basis for further insight into the role of specific bacteria in drug availability within tumors and for understanding the structure-function differences of bacterial and human drug transporters.

Spatial proximity and gene function: a new dimension in prokaryotic gene association network analysis with 3D-GeneNet.

Understanding the biological functions and processes of genes, particularly those not yet characterized, is crucial for advancing molecular biology and identifying therapeutic targets. The hypothesis guiding this study is that the 3D proximity of genes correlates with their functional interactions and relevance in prokaryotes. We introduced 3D-GeneNet, an innovative software tool that utilizes high-throughput sequencing data from chromosome conformation capture techniques and integrates topological metrics to construct gene association networks. Through a series of comparative analyses focused on spatial versus linear distances, we explored various dimensions such as topological structure, functional enrichment levels, distribution patterns of linear distances among gene pairs, and the area under the receiver operating characteristic curve by utilizing model organism Escherichia coli K-12. Furthermore, 3D-GeneNet was shown to maintain good accuracy compared to multiple algorithms (neighbourhood, co-occurrence, coexpression, and fusion) across multiple bacteria, including E. coli, Brucella abortus, and Vibrio cholerae. In addition, the accuracy of 3D-GeneNet's prediction of long-distance gene interactions was identified by bacterial two-hybrid assays on E. coli K-12 MG1655, where 3D-GeneNet not only increased the accuracy of linear genomic distance tripled but also achieved 60% accuracy by running alone. Finally, it can be concluded that the applicability of 3D-GeneNet will extend to various bacterial forms, including Gram-negative, Gram-positive, single-, and multi-chromosomal bacteria through Hi-C sequencing and analysis. Such findings highlight the broad applicability and significant promise of this method in the realm of gene association network. 3D-GeneNet is freely accessible at https://github.com/gaoyuanccc/3D-GeneNet.

Genomic SELEX Screening of Regulatory Targets of Transcription Factors.

The genome of Escherichia coli K-12 is transcribed by a single species of RNA polymerase. The selectivity of transcriptional targets is determined via interaction with one of seven species of the sigma subunit and a total of approximately 300 species of transcription factor (TFs). For comprehensive identification of the regulatory targets of these two groups of regulatory proteins on the genome, we developed an in vitro approach, "Genomic SELEX" (gSELEX) screening. Here we describe a detailed protocol of the gSELEX screening system, which uses purified regulatory proteins and fragments of genomic DNA from E. coli. Moreover, we describe methods and examples of results using cell-free synthetic proteins.

Genetic evidence for functional diversification of gram-negative intermembrane phospholipid transporters.

The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB.

Bactericidal effect of tetracycline in E. coli strain ED1a may be associated with ribosome dysfunction.

Ribosomes translate the genetic code into proteins. Recent technical advances have facilitated in situ structural analyses of ribosome functional states inside eukaryotic cells and the minimal bacterium Mycoplasma. However, such analyses of Gram-negative bacteria are lacking, despite their ribosomes being major antimicrobial drug targets. Here we compare two E. coli strains, a lab E. coli K-12 and human gut isolate E. coli ED1a, for which tetracycline exhibits bacteriostatic and bactericidal action, respectively. Using our approach for close-to-native E. coli sample preparation, we assess the two strains by cryo-ET and visualize their ribosomes at high resolution in situ. Upon tetracycline treatment, these exhibit virtually identical drug binding sites, yet the conformation distribution of ribosomal complexes differs. While K-12 retains ribosomes in a translation-competent state, tRNAs are lost in the vast majority of ED1a ribosomes. These structural findings together with the proteome-wide abundance and thermal stability assessments indicate that antibiotic responses are complex in cells and can differ between different strains of a single species, thus arguing that all relevant bacterial strains should be analyzed in situ when addressing antibiotic mode of action.

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.

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.

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.

Escherichia coli SPFH Membrane Microdomain Proteins HflKC Contribute to Aminoglycoside and Oxidative Stress Tolerance.

Many eukaryotic membrane-dependent functions are often spatially and temporally regulated by membrane microdomains (FMMs), also known as lipid rafts. These domains are enriched in polyisoprenoid lipids and scaffolding proteins belonging to the stomatin, prohibitin, flotillin, and HflK/C (SPFH) protein superfamily that was also identified in Gram-positive bacteria. In contrast, little is still known about FMMs in Gram-negative bacteria. In Escherichia coli K-12, 4 SPFH proteins, YqiK, QmcA, HflK, and HflC, were shown to localize in discrete polar or lateral inner membrane locations, raising the possibility that E. coli SPFH proteins could contribute to the assembly of inner membrane FMMs and the regulation of cellular processes. Here, we studied the determinant of the localization of QmcA and HflC and showed that FMM-associated cardiolipin lipid biosynthesis is required for their native localization pattern. Using Biolog phenotypic arrays, we showed that a mutant lacking all SPFH genes displayed increased sensitivity to aminoglycosides and oxidative stress that is due to the absence of HflKC. Our study therefore provides further insights into the contribution of SPFH proteins to stress tolerance in E. coli. IMPORTANCE Eukaryotic cells often segregate physiological processes in cholesterol-rich functional membrane microdomains. These domains are also called lipid rafts and contain proteins of the stomatin, prohibitin, flotillin, and HflK/C (SPFH) superfamily, which are also present in prokaryotes but have been mostly studied in Gram-positive bacteria. Here, we showed that the cell localization of the SPFH proteins QmcA and HflKC in the Gram-negative bacterium E. coli is altered in the absence of cardiolipin lipid synthesis. This suggests that cardiolipins contribute to E. coli membrane microdomain assembly. Using a broad phenotypic analysis, we also showed that HflKC contribute to E. coli tolerance to aminoglycosides and oxidative stress. Our study, therefore, provides new insights into the cellular processes associated with SPFH proteins in E. coli.

A recombinant strain of Komagataeibacter xylinus ATCC 23770 for production of bacterial cellulose from mannose-rich resources.

The development of bacterial cellulose (BC) industrialization has been seriously affected by its production. Mannose/mannan is an essential component in many biomass resources, but Komagataeibacter xylinus uses mannose in an ineffective way, resulting in waste. The aim of this study was to construct recombinant bacteria to use mannose-rich biomass efficiently as an alternative and inexpensive carbon source in place of the more commonly used glucose. This strategy aimed at modification of the mannose catabolic pathway via genetic engineering of K. xylinus ATCC 23770 strain through expression of mannose kinase and phosphomannose isomerase genes from the Escherichia coli K-12 strain. Recombinant and wild-type strains were cultured under conditions of glucose and mannose respectively as sole carbon sources. The fermentation process and physicochemical properties of BC were investigated in detail in the strains cultured in mannose media. The comparison showed that with mannose as the sole carbon source, the BC yield from the recombinant strain increased by 84%, and its tensile strength and elongation were increased 1.7 fold, while Young's modulus was increased 1.3 fold. The results demonstrated a successful improvement in BC yield and properties on mannose-based medium compared with the wild-type strain. Thus, the strategy of modifying the mannose catabolic pathway of K. xylinus is feasible and has significant potential in reducing the production costs for industrial production of BC from mannose-rich biomass.

4'-Deoxypyridoxine disrupts vitamin B6 homeostasis in Escherichia coli K12 through combined inhibition of cumulative B6 uptake and PLP-dependent enzyme activity.

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6 and a cofactor for many essential metabolic processes such as amino acid biosynthesis and one carbon metabolism. 4'-deoxypyridoxine (4dPN) is a long known B6 antimetabolite but its mechanism of action was not totally clear. By exploring different conditions in which PLP metabolism is affected in the model organism Escherichia coli K12, we showed that 4dPN cannot be used as a source of vitamin B6 as previously claimed and that it is toxic in several conditions where vitamin B6 homeostasis is affected, such as in a B6 auxotroph or in a mutant lacking the recently discovered PLP homeostasis gene, yggS. In addition, we found that 4dPN sensitivity is likely the result of multiple modes of toxicity, including inhibition of PLP-dependent enzyme activity by 4'-deoxypyridoxine phosphate (4dPNP) and inhibition of cumulative pyridoxine (PN) uptake. These toxicities are largely dependent on the phosphorylation of 4dPN by pyridoxal kinase (PdxK).

Evaluation of L-Alanine Metabolism in Bacteria and Whole-Body Distribution with Bacterial Infection Model Mice.

The World Health Organization has cautioned that antimicrobial resistance (AMR) will be responsible for an estimated 10 million deaths annually by 2050. To facilitate prompt and accurate diagnosis and treatment of infectious disease, we investigated the potential of amino acids for use as indicators of bacterial growth activity by clarifying which amino acids are taken up by bacteria during the various growth phases. In addition, we examined the amino acid transport mechanisms that are employed by bacteria based on the accumulation of labeled amino acids, Na+ dependence, and inhibitory effects using a specific inhibitor of system A. We found that 3H-L-Ala accurately reflects the proliferative activity of Escherichia coli K-12 and pathogenic EC-14 in vitro. This accumulation in E. coli could be attributed to the amino acid transport systems being different from those found in human tumor cells. Moreover, biological distribution assessed in infection model mice with EC-14 using 3H-L-Ala showed that the ratio of 3H-L-Ala accumulated in infected muscle to that in control muscle was 1.20. By detecting the growth activity of bacteria in the body that occurs during the early stages of infection by nuclear imaging, such detection methods may result in expeditious diagnostic treatments for infectious diseases.

Phosphatidylglycerol Is the Lipid Donor for Synthesis of Phospholipid-Linked Enterobacterial Common Antigen.

The Gram-negative outer membrane (OM) is an asymmetric bilayer with phospholipids in its inner leaflet and mainly lipopolysaccharide (LPS) in its outer leaflet and is largely impermeable to many antibiotics. In Enterobacterales (e.g., Escherichia, Salmonella, Klebsiella, and Yersinia), the outer leaflet of the OM also contains phosphoglyceride-linked enterobacterial common antigen (ECAPG). This molecule consists of the conserved ECA carbohydrate linked to diacylglycerol-phosphate (DAG-P) through a phosphodiester bond. ECAPG contributes to the OM permeability barrier and modeling suggests that it may alter the packing of LPS molecules in the OM. Here, we investigate, in Escherichia coli K-12, the reaction synthesizing ECAPG from ECA precursor linked to an isoprenoid carrier to identify the lipid donor that provides the DAG-P moiety to ECAPG. Through overexpression of phospholipid biosynthesis genes, we observed alterations expected to increase levels of phosphatidylglycerol (PG) increased the synthesis of ECAPG, whereas alterations expected to decrease levels of PG decreased the synthesis of ECAPG. We discovered depletion of PG levels in strains that could synthesize ECAPG, but not other forms of ECA, causes additional growth defects, likely due to the buildup of ECA precursor on the isoprenoid carrier inhibiting peptidoglycan biosynthesis. Our results demonstrate ECAPG can be synthesized in the absence of the other major phospholipids (phosphatidylethanolamine and cardiolipin). Overall, these results conclusively demonstrate PG is the lipid donor for the synthesis of ECAPG and provide a key insight into the reaction producing ECAPG. In addition, these results provide an interesting parallel to lipoprotein acylation, which also uses PG as its DAG donor. IMPORTANCE The Gram-negative outer membrane is a permeability barrier preventing cellular entry of antibiotics. However, outer membrane biogenesis pathways are targets for small molecule development. Here, we investigate the synthesis of a form of enterobacterial common antigen (ECA), ECAPG, found in the outer membrane of Enterobacterales (e.g., Escherichia, Salmonella, and Klebsiella). ECAPG consists of the conserved ECA carbohydrate unit linked to diacylglycerol-phosphate-ECA is a phospholipid headgroup. The details of the reaction forming this molecule from polymerized ECA precursor are unknown. We determined the lipid donor providing the phospholipid moiety is phosphatidylglycerol. Understanding the synthesis of outer membrane constituents such as ECAPG provides the opportunity for development of molecules to increase outer membrane permeability, expanding the antibiotics available to treat Gram-negative infections.

Dimethyl phthalate inhibits the growth of Escherichia coli K-12 by regulating sugar transport and energy metabolism.

Dimethyl phthalate (DMP) is one of the most widely used plasticizers, and it is easily released into the environment, posing a threat to microbes. In this study, the impact of DMP on the uptake and metabolism of sugars in E. coli K-12 was assessed using proteomics, computational simulation analysis, transcriptome analysis, and sugar utilization experiments. DMP contamination inhibited the growth of E. coli K-12 and downregulated the expression of proteins in ATP-binding cassette (ABC) transporters and the phosphotransferase (PTS) system of E. coli K-12, which are primarily involved in the transmembrane transport of sugars. DMP formed a stable complex with sugar transporters and changed the rigidity and stability of the proteins. Furthermore, DMP treatment decreased the utilization of L-arabinose, glucose, D-xylose, and maltose. Moreover, carbon metabolism and oxidative phosphorylation were also downregulated by DMP. Our study shows that DMP reduces the uptake of sugars and ATP production and subsequently inhibits the growth of E. coli K-12.

Two Trk/Ktr/HKT-type potassium transporters, TrkG and TrkH, perform distinct functions in Escherichia coli K-12.

Escherichia coli K-12 possesses two versions of Trk/Ktr/HKT-type potassium ion (K+) transporters, TrkG and TrkH. The current paradigm is that TrkG and TrkH have largely identical characteristics, and little information is available regarding their functional differences. Here, we show using cation uptake experiments with K+ transporter knockout mutants that TrkG and TrkH have distinct ion transport activities and physiological roles. K+-transport by TrkG required Na+, whereas TrkH-mediated K+ uptake was not affected by Na+. An aspartic acid located five residues away from a critical glycine in the third pore-forming region might be involved in regulation of Na+-dependent activation of TrkG. In addition, we found that TrkG but not TrkH had Na+ uptake activity. Our analysis of K+ transport mutants revealed that TrkH supported cell growth more than TrkG; however, TrkG was able to complement loss of TrkH-mediated K+ uptake in E. coli. Furthermore, we determined that transcription of trkG in E. coli was downregulated but not completely silenced by the xenogeneic silencing factor H-NS (histone-like nucleoid structuring protein or heat-stable nucleoid-structuring protein). Taken together, the transport function of TrkG is clearly distinct from that of TrkH, and TrkG seems to have been accepted by E. coli during evolution as a K+ uptake system that coexists with TrkH.

Transcriptional Potential Determines the Adaptability of Escherichia coli Strains with Different Fitness Backgrounds.

Adaptation through the fitness landscape may be influenced by the gene pool or expression network. However, genetic factors that determine the contribution of beneficial mutations during adaptive evolution are poorly understood. In this study, we experimentally evolved wild-type Escherichia coli K-12 MG1655 and its isogenic derivative that has two additional replication origins and shows higher background fitness. During the short time of experimental evolution, the fitness gains of the two E. coli strains with different fitness backgrounds converged. Populational genome sequencing revealed various mutations with different allele frequencies in evolved populations. Several mutations occurred in genes affecting transcriptional regulation (e.g., RNA polymerase subunit, RNase, ppGpp synthetase, and transcription termination/antitermination factor genes). When we introduced mutations into the ancestral E. coli strains, beneficial effects tended to be lower in the ancestor with higher initial fitness. Replication rate analysis showed that the various replication indices do not correlate with the growth rate. Transcriptome profiling showed that gene expression and gene ontology are markedly enriched in populations with lower background fitness after experimental evolution. Further, the degree of transcriptional change was proportional to the fitness gain. Thus, the evolutionary trajectories of bacteria with different fitness backgrounds can be complex and counterintuitive. Notably, transcriptional change is a major contributor to adaptability. IMPORTANCE Predicting the adaptive potential of bacterial populations can be difficult due to their complexity and dynamic environmental conditions. Also, epistatic interaction between mutations affects the adaptive trajectory. Nevertheless, next-generation sequencing sheds light on understanding evolutionary dynamics through high-throughput genome and transcriptome information. Experimental evolution of two E. coli strains with different background fitness showed that the trajectories of fitness gain, which slowed down during the later stages of evolution, became convergent. This suggests that the adaptability of bacteria can be counterintuitive and that predicting the evolutionary path of bacteria can be difficult even in a constant environment. In addition, transcriptional change is associated with fitness gain during the evolutionary process. Thus, the adaptability of cells depends on their intrinsic genetic capacity for a given evolutionary period. This should be considered when genetically engineered bacteria are optimized through adaptive evolution.

The TonB-dependent uptake of pyrroloquinoline-quinone (PQQ) and secretion of gluconate by Escherichia coli K-12.

Glucose is taken up by Escherichia coli through the phosphotransferase system (PTS) as the preferred carbon source. PTS mutants grow with glucose as a carbon source only in the presence of pyrroloquinoline quinone (PQQ), which is needed as a redox cofactor for the glucose dehydrogenase Gcd. The membrane-anchored Gcd enzyme oxidises glucose to gluconolactone in the periplasm. For this reaction to occur, external supply of PQQ is required as E. coli is unable to produce PQQ de novo. Growth experiments show that PqqU (previously YncD) is the TonB-ExbBD-dependent transporter for PQQ through the outer membrane. PQQ protected the cells from the PqqU-dependent phage IsaakIselin (Bas10) by competition for the receptor protein. As a high affinity uptake system, PqqU allows E. coli to activate Gcd even at surrounding PQQ concentrations of about 1 nmoL/L. At about 30-fold higher PQQ concentrations, the activation of Gcd gets PqqU independent. Due to its small size, Pqq may also pass the outer membrane through porins. The PQQ-dependent production of gluconate has been demonstrated in many plant growth-promoting bacteria that solubilise phosphate minerals in the soil by secreting this acid. Under phosphate limiting conditions also E. coli induces the glucose dehydrogenase and secretes gluconate, even in absence of PTS, that is, even when the bacterium is unable to grow on glucose without PQQ.

The absence of the queuosine tRNA modification leads to pleiotropic phenotypes revealing perturbations of metal and oxidative stress homeostasis in Escherichia coli K12.

Queuosine (Q) is a conserved hypermodification of the wobble base of tRNA containing GUN anticodons but the physiological consequences of Q deficiency are poorly understood in bacteria. This work combines transcriptomic, proteomic and physiological studies to characterize a Q-deficient Escherichia coli K12 MG1655 mutant. The absence of Q led to an increased resistance to nickel and cobalt, and to an increased sensitivity to cadmium, compared to the wild-type (WT) strain. Transcriptomic analysis of the WT and Q-deficient strains, grown in the presence and absence of nickel, revealed that the nickel transporter genes (nikABCDE) are downregulated in the Q- mutant, even when nickel is not added. This mutant is therefore primed to resist to high nickel levels. Downstream analysis of the transcriptomic data suggested that the absence of Q triggers an atypical oxidative stress response, confirmed by the detection of slightly elevated reactive oxygen species (ROS) levels in the mutant, increased sensitivity to hydrogen peroxide and paraquat, and a subtle growth phenotype in a strain prone to accumulation of ROS.

Transcription Factor SrsR (YgfI) Is a Novel Regulator for the Stress-Response Genes in Stationary Phase in Escherichia coli K-12.

Understanding the functional information of all genes and the biological mechanism based on the comprehensive genome regulation mechanism is an important task in life science. YgfI is an uncharacterized LysR family transcription factor in Escherichia coli. To identify the function of YgfI, the genomic SELEX (gSELEX) screening was performed for YgfI regulation targets on the E. coli genome. In addition, regulatory and phenotypic analyses were performed. A total of 10 loci on the E. coli genome were identified as the regulatory targets of YgfI with the YgfI binding activity. These predicted YgfI target genes were involved in biofilm formation, hydrogen peroxide resistance, and antibiotic resistance, many of which were expressed in the stationary phase. The TCAGATTTTGC sequence was identified as an YgfI box in in vitro gel shift assay and DNase-I footprinting assays. RT-qPCR analysis in vivo revealed that the expression of YgfI increased in the stationary phase. Physiological analyses suggested the participation of YgfI in biofilm formation and an increase in the tolerability against hydrogen peroxide. In summary, we propose to rename ygfI as srsR (a stress-response regulator in stationary phase).