The construction of an engineered bacterium to remove cadmium from wastewater

2014 ◽  
Vol 70 (12) ◽  
pp. 2015-2021 ◽  
Author(s):  
S. Chang ◽  
H. Shu
Keyword(s):  
Phytochelatin Synthase ◽  
Serine Acetyltransferase ◽  
Cd Accumulation ◽  
Glutathione Synthesis ◽  
E Coli ◽  
Limiting Step ◽  
Atpase Gene ◽  
Key Genes ◽  
Engineered Bacteria ◽  
Heavy Metal Atpase

The removal of cadmium (Cd) from wastewater before it is released from factories is important for protecting human health. Although some researchers have developed engineered bacteria, the resistance of these engineered bacteria to Cd have not been improved. In this study, two key genes involved in glutathione synthesis (gshA and gshB), a serine acetyltransferase gene (cysE), a Thlaspi caerulescens phytochelatin synthase gene (TcPCS1), and a heavy metal ATPase gene (TcHMA3) were transformed into Escherichia coli BL21. The resistance of the engineered bacterium to Cd was significantly greater than that of the initial bacterium and the Cd accumulation in the engineered bacterium was much higher than in the initial bacterium. In addition, the Cd resistance of the bacteria harboring gshB, gshA, cysE, and TcPCS1 was higher than that of the bacteria harboring gshA, cysE, and TcPCS1. This finding demonstrated that gshB played an important role in glutathione synthesis and that the reaction catalyzed by glutathione synthase was the limiting step for producing phytochelatins. Furthermore, TcPCS1 had a greater specificity and a higher capacity for removing Cd than SpPCS1, and TcHMA3 not only played a role in T. caerulescens but also functioned in E. coli.

scholarly journals Enhanced Extracellular Synthesis of Gold Nanoparticles by Soluble Extracts from Escherichia coli Transformed with Rhizobium tropici Phytochelatin Synthase Gene

Metals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 472
Author(s):  
Qunying Yuan ◽  
Manjula Bomma ◽  
Zhigang Xiao
Keyword(s):  
Gold Nanoparticles ◽  
Gold Nanoparticle ◽  
Synthesis Method ◽  
Nanoparticle Synthesis ◽  
Morphological Properties ◽  
Phytochelatin Synthase ◽  
Rhizobium Tropici ◽  
Whole Cells ◽  
E Coli ◽  
Recombinant Cells

Phytochelatins, the enzymatic products of phytochelatin synthase, play a principal role in protecting the plants from heavy metal and metalloid toxicity due to their ability to scavenge metal ions. In the present study, we investigated the capacity of soluble intracellular extracts from E. coli cells expressing R. tropici phytochelatin synthase to synthesize gold nanoparticle. We discovered that the reaction mediated by soluble extracts from the recombinant E. coli cells had a higher yield of gold nanoparticles, compared to that from the control cells. The compositional and morphological properties of the gold nanoparticles synthesized by the intracellular extracts from recombinant cells and control cells were similar. In addition, this extracellular nanoparticle synthesis method produced purer gold nanoparticles, avoiding the isolation of nanoparticles from cellular debris when whole cells are used to synthesize nanoparticles. Our results suggested that phytochelatins can improve the efficiency of gold nanoparticle synthesis mediated by bacterial soluble intracellular extracts, and the potential of extracellular nanoparticle synthesis platform for the production of nanoparticles in large quantity and pure form is worth further investigation.

scholarly journals Metabolome and proteome analyses reveal transcriptional misregulation in glycolysis of engineered E. coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chun-Ying Wang ◽  
Martin Lempp ◽  
Niklas Farke ◽  
Stefano Donati ◽  
Timo Glatter ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Metabolic Pathways ◽  
Glycerol Production ◽  
Inducible Promoters ◽  
E Coli ◽  
Inhibition Of Glycolysis ◽  
Underlying Mechanisms ◽  
Rate Limiting ◽  
Engineered Bacteria

AbstractSynthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Here we show that the misregulated activity of the transcription factor Cra is responsible for the growth burden of glycerol overproducing E. coli. Glycerol production decreases the concentration of fructose-1,6-bisphoshate (FBP), which then activates Cra resulting in the downregulation of glycolytic enzymes and upregulation of gluconeogenesis enzymes. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway. We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. We show the broad applicability of this approach by engineering Cra-dependent regulation into a set of constitutive and inducible promoters, and use one of them to overproduce carotenoids in E. coli.

scholarly journals Encrypting messages with artificial bacterial receptors

2020 ◽  
Vol 16 ◽  
pp. 2749-2756
Author(s):  
Pragati Kishore Prasad ◽  
Naama Lahav-Mankovski ◽  
Leila Motiei ◽  
David Margulies
Keyword(s):  
Protein C ◽  
Molecular Level ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Potential Contribution ◽  
Information Protection ◽  
Emission Pattern ◽  
E Coli ◽  
Artificial Receptors ◽  
Engineered Bacteria

A method for encrypting messages using engineered bacteria and different fluorescently labeled synthetic receptors is described. We show that the binding of DNA-based artificial receptors to E. coli expressing His-tagged outer membrane protein C (His-OmpC) induces a Förster resonance energy transfer (FRET) between the dyes, which results in the generation of a unique fluorescence fingerprint. Because the bacteria continuously divide, the emission pattern generated by the modified bacteria dynamically changes, enabling the system to produce encryption keys that change with time. Thus, this development indicates the potential contribution of live-cell-based encryption systems to the emerging area of information protection at the molecular level.

scholarly journals Analysis of weighted gene co-expression network of triterpenoid-related transcriptome characteristics from different strains of Wolfiporia cocos

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Guiping Zeng ◽  
Zhong Li ◽  
Zhi Zhao
Keyword(s):  
Chinese Medicine ◽  
Target Gene ◽  
Pharmacological Effects ◽  
Gene Modification ◽  
Medicinal Value ◽  
Key Genes ◽  
Different Strains ◽  
Engineered Bacteria ◽  
Core Genes

AbstractThe fungus Wolfiporia cocos has wide-ranging and important medicinal value, and its dried sclerotia are used as a traditional Chinese medicine. Modern studies have shown that triterpenoid, the active ingredient of W. cocos, have a variety of pharmacological effects. The aim of our research was to determine the key genes related to triterpenoid biosynthesis, which may be useful for the genetic modification of cell-engineered bacteria for triterpenoid biosynthesis. In this study, two monospore strains, DZAC-WP-H-29 (high-yielding) and DZAC-WP-L-123 (low-yielding), were selected from the sexually propagated offspring of strain 5.78 of W. cocos, and the mycelia were cultured for 17, 34, and 51 days, respectively. Weighted gene co-expression network analysis (WGCNA) method was used to analyze transcriptional expressions. The results show that eight core genes (ACAT1-b, hgsA, mvd1, SQLE, erg6, TAT, erg26, and erg11) are associated with the triterpenoid synthesis pathway, and Pm20d2 and norA outside the pathway may be important genes that influence the biosynthesis and accumulation of W. cocos triterpenoid. The biosynthesis of W. cocos triterpenoid is closely related to the expression of sterol metabolic pathway genes. The role of these genes in triterpenoid synthesis complements our knowledge on the biosynthesis and accumulation of W. cocos triterpenoid, and also provides a reference for the target gene modification of engineered bacteria for the fermentation production of triterpenoid.

scholarly journals CRISPR-Cas Controls Cryptic Prophages

2021 ◽  
Author(s):  
Sooyeon Song ◽  
Thomas K Wood
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Adaptive Immune System ◽  
The Novel ◽  
E Coli ◽  
Adaptive Immune ◽  
Persister Cell ◽  
Key Genes ◽  
K 12 ◽  
Cas Proteins

The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be non-functional in the best-studied bacterium, Escherichia coli K-12. Instead, we demonstrate here that the E. coli CRISPR-Cas system is active and inhibits its nine defective (i.e., cryptic) prophages. Specifically, deactivation of CRISPR-Cas via deletion of cas2, which encodes one of the two conserved CRISPR-Cas proteins, reduces growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation; hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages. Consistently, seven of the 13 E. coli spacers contain matches to the cryptic prophages, and, after excision, CRISPR-Cas cleaves cryptic prophage CP4-57 and DLP-12 DNA. Moreover, we determine that the key genes in these cryptic prophages that CRISPR-Cas represses by cleaving the excised DNA include lysis protein YdfD of Qin and lysis protein RzoD of DLP-12. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages; i.e., unlike with active lysogens, CRISPR-Cas and cryptic prophages may stably exist.

scholarly journals Variation in the BrHMA3 coding region controls natural variation in cadmium accumulation in Brassica rapa vegetables

2019 ◽  
Vol 70 (20) ◽  
pp. 5865-5878 ◽  
Author(s):  
Lingxiao Zhang ◽  
Jian Wu ◽  
Zhong Tang ◽  
Xin-Yuan Huang ◽  
Xiaowu Wang ◽  
...  
Keyword(s):  
Brassica Rapa ◽  
Promoter Sequence ◽  
Full Length ◽  
Vegetable Crops ◽  
Coding Region ◽  
Cd Uptake ◽  
Cd Accumulation ◽  
Coding Sequence ◽  
Atpase Gene ◽  
Cd Translocation

Abstract Brassica rapa includes several important leafy vegetable crops with the potential for high cadmium (Cd) accumulation, posing a risk to human health. This study aims to understand the genetic basis underlying the variation in Cd accumulation among B. rapa vegetables. Cd uptake and translocation in 64 B. rapa accessions were compared. The role of the heavy metal ATPase gene BrHMA3 in the variation of Cd accumulation was investigated. BrHMA3 encodes a tonoplast-localized Cd transporter. Five full-length and four truncated haplotypes of the BrHMA3 coding sequence were identified, explaining >80% of the variation in the Cd root to shoot translocation among the 64 accessions and in F2 progeny. Truncated BrHMA3 haplotypes had a 2.3 and 9.3 times higher shoot Cd concentration and Cd translocation ratio, respectively, than full-length haplotypes. When expressed in yeast and Arabidopsis thaliana, full-length BrHMA3 showed activity consistent with a Cd transport function, whereas truncated BrHMA3 did not. Variation in the BrHMA3 promoter sequence had little effect on Cd translocation. Variation in the BrHMA3 coding sequence is a key determinant of Cd translocation to and accumulation in the leaves of B. rapa. Strong alleles of BrHMA3 can be used to breed for B. rapa vegetables that are low in Cd in their edible portions.

scholarly journals Threshold accumulation of a constitutive protein explains E. coli cell-division behavior in nutrient upshifts

2021 ◽  
Vol 118 (18) ◽  
pp. e2016391118
Author(s):  
Mia Panlilio ◽  
Jacopo Grilli ◽  
Giorgio Tallarico ◽  
Ilaria Iuliani ◽  
Bianca Sclavi ◽  
...  
Keyword(s):  
Cell Division ◽  
Recent Progress ◽  
Single Cells ◽  
Growth Behavior ◽  
Division Rate ◽  
E Coli ◽  
Limiting Step ◽  
Size Growth ◽  
Molecular Origin ◽  
Insight Into

Despite a boost of recent progress in dynamic single-cell measurements and analyses in Escherichia coli, we still lack a mechanistic understanding of the determinants of the decision to divide. Specifically, the debate is open regarding the processes linking growth and chromosome replication to division and on the molecular origin of the observed “adder correlations,” whereby cells divide, adding roughly a constant volume independent of their initial volume. In order to gain insight into these questions, we interrogate dynamic size-growth behavior of single cells across nutrient upshifts with a high-precision microfluidic device. We find that the division rate changes quickly after nutrients change, much before growth rate goes to a steady state, and in a way that adder correlations are robustly conserved. Comparison of these data to simple mathematical models falsifies proposed mechanisms, where replication–segregation or septum completions are the limiting step for cell division. Instead, we show that the accumulation of a putative constitutively expressed “P-sector divisor” protein explains the behavior during the shift.

scholarly journals Role of AmpC-Inducing Genes in Modulating Other Serine Beta-Lactamases in Escherichia coli

Antibiotics ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 67
Author(s):  
Dhriti Mallik ◽  
Diamond Jain ◽  
Sanjib Bhakta ◽  
Anindya Sundar Ghosh
Keyword(s):  
Negative Regulator ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
E Coli ◽  
Global Challenge ◽  
Induction Mechanism ◽  
In Trans ◽  
Key Genes

The consistently mutating bacterial genotypes appear to have accelerated the global challenge with antimicrobial resistance (AMR); it is therefore timely to investigate certain less-explored fields of targeting AMR mechanisms in bacterial pathogens. One of such areas is beta-lactamase (BLA) induction that can provide us with a collection of prospective therapeutic targets. The key genes (ampD, ampE and ampG) to which the AmpC induction mechanism is linked are also involved in regulating the production of fragmented muropeptides generated during cell-wall peptidoglycan recycling. Although the involvement of these genes in inducing class C BLAs is apparent, their effect on serine beta-lactamase (serine-BLA) induction is little known. Here, by using ∆ampD and ∆ampE mutants of E. coli, we attempted to elucidate the effects of ampD and ampE on the expression of serine-BLAs originating from Enterobacteriaceae, viz., CTX-M-15, TEM-1 and OXA-2. Results show that cefotaxime is the preferred inducer for CTX-M-15 and amoxicillin for TEM-1, whereas oxacillin for OXA-2. Surprisingly, exogenous BLA expressions are elevated in ∆ampD and ∆ampE mutants but do not always alter their beta-lactam susceptibility. Moreover, the beta-lactam resistance is increased upon in trans expression of ampD, whereas the same is decreased upon ampE expression, indicating a differential effect of ampD and ampE overexpression. In a nutshell, depending on the BLA, AmpD amidase moderately facilitates a varying level of serine-BLA expression whereas AmpE transporter acts likely as a negative regulator of serine-BLA.

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