Effects of the Expression of Random Sequence Clones on Growth and Transcriptome Regulation in Escherichia coli

Comparative genomic analyses have provided evidence that new genetic functions can emerge out of random nucleotide sequences. Here, we apply a direct experimental approach to study the effects of plasmids harboring random sequence inserts under the control of an inducible promoter. Based on data from previously described experiments dealing with the growth of clones within whole libraries, we extracted specific clones that had shown either negative, neutral or positive effects on relative cell growth. We analyzed these individually with respect to growth characteristics and the impact on the transcriptome. We find that candidate clones for negative peptides lead to growth arrest by eliciting a general stress response. Overexpression of positive clones, on the other hand, does not change the exponential growth rates of hosts, and they show a growth advantage over a neutral clone when tested in direct competition experiments. Transcriptomic changes in positive clones are relatively moderate and specific to each clone. We conclude from our experiments that random sequence peptides are indeed a suitable source for the de novo evolution of genetic functions.

The MocR/GabR Ectoine and Hydroxyectoine Catabolism Regulator EnuR: Inducer and DNA Binding

The compatible solutes ectoine and 5-hydroxyectoine are widely synthesized by bacteria as osmostress protectants. These nitrogen-rich tetrahydropyrimidines can also be exploited as nutrients by microorganisms. Many ectoine/5-hydroxyectoine catabolic gene clusters are associated with a regulatory gene (enuR: ectoine nutrient utilization regulator) encoding a repressor protein belonging to the MocR/GabR sub-family of GntR-type transcription factors. Focusing on EnuR from the marine bacterium Ruegeria pomeroyi, we show that the dimerization of EnuR is mediated by its aminotransferase domain. This domain can fold independently from its amino-terminal DNA reading head and can incorporate pyridoxal-5′-phosphate (PLP) as cofactor. The covalent attachment of PLP to residue Lys302 of EnuR was proven by mass-spectrometry. PLP interacts with system-specific, ectoine and 5-hydroxyectoine-derived inducers: alpha-acetyldiaminobutyric acid (alpha-ADABA), and hydroxy-alpha-acetyldiaminobutyric acid (hydroxy-alpha-ADABA), respectively. These inducers are generated in cells actively growing with ectoines as sole carbon and nitrogen sources, by the EutD hydrolase and targeted metabolic analysis allowed their detection. EnuR binds these effector molecules with affinities in the low micro-molar range. Studies addressing the evolutionary conservation of EnuR, modelling of the EnuR structure, and docking experiments with the inducers provide an initial view into the cofactor and effector binding cavity. In this cavity, the two high-affinity inducers for EnuR, alpha-ADABA and hydroxy-alpha-ADABA, are positioned such that their respective primary nitrogen group can chemically interact with PLP. Purified EnuR bound with micro-molar affinity to a 48 base pair DNA fragment containing the sigma-70 type substrate-inducible promoter for the ectoine/5-hydroxyectoine importer and catabolic gene cluster. Consistent with the function of EnuR as a repressor, the core elements of the promoter overlap with two predicted EnuR operators. Our data lend themselves to a straightforward regulatory model for the initial encounter of EnuR-possessing ectoine/5-hydroxyectoine consumers with environmental ectoines and for the situation when the external supply of these compounds has been exhausted by catabolism.

Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells

Human adenoviruses (HAdV) cause a variety of infections in human hosts, from self-limited upper respiratory tract infections in otherwise healthy people to fulminant pneumonia and death in immunocompromised patients. Many HAdV enter polarized epithelial cells by using the primary receptor, the Coxsackievirus and adenovirus receptor (CAR). Recently published data demonstrate that a potent neutrophil (PMN) chemoattractant, interleukin-8 (IL-8), stimulates airway epithelial cells to increase expression of the apical isoform of CAR (CAREx8), which results in increased epithelial HAdV type 5 (HAdV5) infection. However, the mechanism for PMN-enhanced epithelial HAdV5 transduction remains unclear. In this manuscript, the molecular mechanisms behind PMN mediated enhancement of epithelial HAdV5 transduction are characterized using an MDCK cell line that stably expresses human CAREx8 under a doxycycline inducible promoter (MDCK-CAREx8 cells). Contrary to our hypothesis, PMN exposure does not enhance HAdV5 entry by increasing CAREx8 expression nor through activation of non-specific epithelial endocytic pathways. Instead, PMN serine proteases are responsible for PMN-mediated enhancement of HAdV5 transduction in MDCK-CAREx8 cells. This is evidenced by reduced transduction upon inhibition of PMN serine proteases and increased transduction upon exposure to exogenous human neutrophil elastase (HNE). Furthermore, HNE exposure activates epithelial autophagic flux, which, even when triggered through other mechanisms, results in a similar enhancement of epithelial HAdV5 transduction. Inhibition of F-actin with cytochalasin D partially attenuates PMN mediated enhancement of HAdV transduction. Taken together, these findings suggest that HAdV5 can leverage innate immune responses to establish infections.

Chemical biology-whole genome engineering datasets predict new antibacterial combinations

Trimethoprim and sulfamethoxazole are used commonly together as cotrimoxazole for the treatment of urinary tract and other infections. The evolution of resistance to these and other antibacterials threatens therapeutic options for clinicians. We generated and analysed a chemical-biology-whole-genome data set to predict new targets for antibacterial combinations with trimethoprim and sulfamethoxazole. For this we used a large transposon mutant library in Escherichia coli BW25113 where an outward-transcribing inducible promoter was engineered into one end of the transposon. This approach allows regulated expression of adjacent genes in addition to gene inactivation at transposon insertion sites, a methodology that has been called TraDIS-Xpress. These chemical genomic data sets identified mechanisms for both reduced and increased susceptibility to trimethoprim and sulfamethoxazole. The data identified that over-expression of FolA reduced trimethoprim susceptibility, a known mechanism for reduced susceptibility. In addition, transposon insertions into the genes tdk, deoR, ybbC, hha, ldcA, wbbK and waaS increased susceptibility to trimethoprim and likewise for rsmH, fadR, ddlB, nlpI and prc with sulfamethoxazole, while insertions in ispD, uspC, minC, minD, yebK, truD and umpG increased susceptibility to both these antibiotics. Two of these genes’ products, Tdk and IspD, are inhibited by AZT and fosmidomycin respectively, antibiotics that are known to synergise with trimethoprim. Thus, the data identified two known targets and several new target candidates for the development of co-drugs that synergise with trimethoprim, sulfamethoxazole or cotrimoxazole. We demonstrate that the TraDIS-Xpress technology can be used to generate information-rich chemical-genomic data sets that can be used for antibacterial development.

Effects of the expression of random sequence clones on growth and transcriptome regulation in Escherichia coli

Comparative genomic analyses have provided evidence that new genetic functions can emerge out of random nucleotide sequences. Here, we apply a direct experimental approach to study the effects of plasmids harbouring random sequence inserts under the control of an inducible promoter. Based on data from previously described experiments dealing with the growth of clones within whole libraries, we extracted specific clones that had shown either negative or positive effects on relative cell growth. We analysed these individually with respect to growth characteristics and impact on the transcriptome. We find that candidate clones for negative peptides lead to growth arrest by eliciting a general stress response. Overexpression of positive clones, on the other hand, does not change the exponential growth rates of hosts, but they show a growth advantage over a neutral candidate clone when tested in direct competition experiments. Transcriptomic changes in positive clones are relatively moderate and specific to each clone. We conclude from our experiments that random sequence peptides are in-deed a suitable source for the de novo evolution of genetic functions.

A Myo-Inositol-Inducible Expression System for Corynebacterium glutamicum and Its Application

Corynebacterium glutamicum is one of the important industrial microorganisms for production of amino acids and other value-added compounds. Most expression vectors used in C. glutamicum are based on inducible promoter (Ptac or Ptrc) activated by isopropyl-β-D-thiogalactopyranoside (IPTG). However, these vectors seem unsuitable for large-scale industrial production due to the high cost and toxicity of IPTG. Myo-inositol is an ideal inducer because of its non-toxicity and lower price. In this study, a myo-inositol-inducible expression vector pMI-4, derived from the expression vector pXMJ19, was constructed. Besides the original chloramphenicol resistance gene cat, multiple cloning sites, and rrnB terminator, the pMI-4 (6,643 bp) contains the iolRq cassette and the myo-inositol-inducible promoter PiolT1. The pMI-4 could stably replicate in the C. glutamicum host. Meanwhile, the non-myo-inositol degradation host strain C. glutamicumΔiolGΔoxiCΔoxiDΔoxiE for maintaining the pMI-4 was developed. Overexpression of hemAM and hemL using pMI-4 resulted in a significant accumulation of 5-aminolevulinic acid, indicating its potential application in metabolic engineering and industrial fermentation.

A Recombinant Adenoviral Vector With a Specific Tropism to CD4-positive Cells: a New Tool for HIV-1 Inhibition

Gene therapy can be an option to overcoming the side effects of chemotherapy and preventing the development of drug-resistant HIV viruses in HIV-infected patients. The need to develop a safe and efficient vector for gene transfer is always necessary and an appropriate option might be adenovirus (Ad). the use of Ad vectors in the gene delivery applications is limited due to the semi-specific tropism. A strategy to overcome this tropism limitation may be the modification of fiber protein domain involved in the viral binding to cells. Therefore, we designed an Ad5 vector with a specific tropism to CD4+ cells containing an expression system limited to HIV-infected cells. We replaced the knob region of Ad5 fiber protein with the extracellular region of HIV-1 envelope. We also used a specific Tat-inducible promoter to express two anti-HIV-1 shRNAs. Tropism of recombinant Ad5 was assayed by comparison of shRNA expression level in CEM and PBMC cells (as CD4+ cells) and HEK293 cells (as CD4- cells). HIV-1 inhibition was assayed by determination of p24 antigen in the HIV-infected CEM cells transduced with the recombinant Ad5 vector. Our results showed that shRNA expression was significantly higher in CEM and PBMC cells than HEK293 cells when were transduced with recombinant Ad5 vector. This new Ad5 vector also inhibited HIV-1 proliferation in a Tat-inducible manner. Our new recombinant Ad5 vector has a specific tropism to CD4-positive cells that can effectively suppress the HIV-1 replication.

Application and Construction of Microbial Biosensors in Chemical Forensics

<p>Forensic toxicologists are often required to rapidly determine if a suspicious substance, such as a white powder, contain toxins. Preliminary tests usually include screens for a wide range of 'Potentially Toxic Chemicals' (PTCs) such as cyanide, pesticides, herbicides, medicinal and illicit drugs. Subsequent analyses are generally very time-consuming and costly. Any protocol screening for a range of PTC's, prior to more robust chemical analysis, could therefore save significant analytical time. Microbial biosensors are ideal biological tools that can be utilised for these purposes. In vivo bioassays were developed for a range of PTCs using a suite of microbial biosensors, in a variety of complex matrices including water, white powders, soils and vomit to determine the effect of matrix complexities on the biosensors, as well as the toxins. The lux biosensor, Escherichia coli HB101 pUCD607, showed an EC50, (where EC50 is the effective concentration of toxin causing 50% reduction in bioluminescence), of cyanide in water of 20 mg/L. This biosensor still detected cyanide, in talc and flour, at EC50 values of 589 mg/L and 700 mg/L respectively. Vibrio harveyi showed good sensitivity to cyanide in initial water bioassays with an EC50 of 9.66 mg/L. The V. harveyi biosensor did not detect cyanide spiked in talc or flour when tested up to a maximum concentration of 10,000 mg/L. The Mycena citricolor ATCC 34884 fungal biosensor, showed lower sensitivity levels however it detected the presence of sodium monofluoroacetate (1080) at a concentration 1000 mg/L. Preliminary investigation of a novel, faster, solid-phase sample preparation method was also undertaken and its potential proven, particularly in PTC spiked white powders. Here the biosensor showed sensitivity to arsenate, arsenite, copper, cyanide and PCP at 1000 mg/L.This project highlighted the inability of current biosensors to reliably detect 1080 and the difficulty in constructing a specific biosensor. The utilisation of a reliable vector and inducible promoter are pivotal in biosensor construction.</p>

Application and Construction of Microbial Biosensors in Chemical Forensics

<p>Forensic toxicologists are often required to rapidly determine if a suspicious substance, such as a white powder, contain toxins. Preliminary tests usually include screens for a wide range of 'Potentially Toxic Chemicals' (PTCs) such as cyanide, pesticides, herbicides, medicinal and illicit drugs. Subsequent analyses are generally very time-consuming and costly. Any protocol screening for a range of PTC's, prior to more robust chemical analysis, could therefore save significant analytical time. Microbial biosensors are ideal biological tools that can be utilised for these purposes. In vivo bioassays were developed for a range of PTCs using a suite of microbial biosensors, in a variety of complex matrices including water, white powders, soils and vomit to determine the effect of matrix complexities on the biosensors, as well as the toxins. The lux biosensor, Escherichia coli HB101 pUCD607, showed an EC50, (where EC50 is the effective concentration of toxin causing 50% reduction in bioluminescence), of cyanide in water of 20 mg/L. This biosensor still detected cyanide, in talc and flour, at EC50 values of 589 mg/L and 700 mg/L respectively. Vibrio harveyi showed good sensitivity to cyanide in initial water bioassays with an EC50 of 9.66 mg/L. The V. harveyi biosensor did not detect cyanide spiked in talc or flour when tested up to a maximum concentration of 10,000 mg/L. The Mycena citricolor ATCC 34884 fungal biosensor, showed lower sensitivity levels however it detected the presence of sodium monofluoroacetate (1080) at a concentration 1000 mg/L. Preliminary investigation of a novel, faster, solid-phase sample preparation method was also undertaken and its potential proven, particularly in PTC spiked white powders. Here the biosensor showed sensitivity to arsenate, arsenite, copper, cyanide and PCP at 1000 mg/L.This project highlighted the inability of current biosensors to reliably detect 1080 and the difficulty in constructing a specific biosensor. The utilisation of a reliable vector and inducible promoter are pivotal in biosensor construction.</p>

The HrpX Protein Activates Synthesis of the RaxX Sulfopeptide, Required for Activation of XA21-Mediated Immunity to Xanthomonas oryzae pv. oryzae

Upon encountering a susceptible plant host, a bacterial pathogen expresses specific virulence factors. For example, in planta, the Xanthomonas HrpX protein activates transcription of roughly 150 genes encoding components of the type III secretion system or its translocated effectors, as well as other secreted proteins implicated in pathogenesis. Here, we show that X. oryzae pv. oryzae growth in planta or in HrpX-inducing XOM2 media resulted in HrpX-dependent transcription of the raxX and raxST genes that control production of the RaxX sulfopeptide, exported through a type I secretion system. The RaxX protein is required for activation of XA21-mediated immunity in Xa21+ rice lines. We identified potential plant-inducible promoter elements upstream of the likely 5′ ends of the raxX and raxST transcripts. Deletions and nucleotide substitutions confirmed that these elements are required for HrpX-dependent expression of raxX and raxST. We conclude that raxX-raxST gene expression is induced by HrpX during growth in planta and, therefore, is coordinately expressed with other genes required for pathogenesis. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .