scholarly journals Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Kyle A. Zingaro ◽  
Eleftherios Terry Papoutsakis
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Solvent Tolerance ◽  
Content Type ◽  
Response System ◽  
Wide Range ◽  
Stress Response System ◽  
Shock Proteins

ABSTRACT Strain tolerance to toxic metabolites is an important trait for many biotechnological applications, such as the production of solvents as biofuels or commodity chemicals. Engineering a complex cellular phenotype, such as solvent tolerance, requires the coordinated and tuned expression of several genes. Using combinations of heat shock proteins (HSPs), we engineered a semisynthetic stress response system in Escherichia coli capable of tolerating high levels of toxic solvents. Simultaneous overexpression of the HSPs GrpE and GroESL resulted in a 2-fold increase in viable cells (CFU) after exposure to 5% (vol/vol) ethanol for 24 h. Co-overexpression of GroESL and ClpB on coexisting plasmids resulted in 1,130%, 78%, and 25% increases in CFU after 24 h in 5% ethanol, 1% n-butanol, and 1% i-butanol, respectively. Co-overexpression of GrpE, GroESL, and ClpB on a single plasmid produced 200%, 390%, and 78% increases in CFU after 24 h in 7% ethanol, 1% n-butanol, or 25% 1,2,4-butanetriol, respectively. Overexpression of other autologous HSPs (DnaK, DnaJ, IbpA, and IbpB) alone or in combinations failed to improve tolerance. Expression levels of HSP genes, tuned through inducible promoters and the plasmid copy number, affected the effectiveness of the engineered stress response system. Taken together, these data demonstrate that tuned co-overexpression of GroES, GroEL, ClpB, and GrpE can be engaged to engineer a semisynthetic stress response system capable of greatly increasing the tolerance of E. coli to solvents and provides a starting platform for engineering customized tolerance to a wide variety of toxic chemicals. IMPORTANCE Microbial production of useful chemicals is often limited by the toxicity of desired products, feedstock impurities, and undesired side products. Improving tolerance is an essential step in the development of practical platform organisms for production of a wide range of chemicals. By overexpressing autologous heat shock proteins in Escherichia coli, we have developed a modular semisynthetic stress response system capable of improving tolerance to ethanol, n-butanol, and potentially other toxic solvents. Using this system, we demonstrate that a practical stress response system requires both tuning of individual gene components and a reliable framework for gene expression. This system can be used to seek out new interacting partners to improve the tolerance phenotype and can be used in the development of more robust solvent production strains.

scholarly journals Functional Analysis of Genes Comprising the Locus of Heat Resistance in Escherichia coli

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Ryan Mercer ◽  
Oanh Nguyen ◽  
Qixing Ou ◽  
Lynn McMullen ◽  
Michael G. Gänzle
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Heat Resistance ◽  
Growth Phase ◽  
Genomic Island ◽  
Enteric Bacteria ◽  
Content Type ◽  
E Coli ◽  
Shock Proteins

ABSTRACT The locus of heat resistance (LHR) is a 15- to 19-kb genomic island conferring exceptional heat resistance to organisms in the family Enterobacteriaceae, including pathogenic strains of Salmonella enterica and Escherichia coli. The complement of LHR-comprising genes that is necessary for heat resistance and the stress-induced or growth-phase-induced expression of LHR-comprising genes are unknown. This study determined the contribution of the seven LHR-comprising genes yfdX1 GI, yfdX2, hdeD GI, orf11, trx GI, kefB, and psiE GI by comparing the heat resistances of E. coli strains harboring plasmid-encoded derivatives of the different LHRs in these genes. (Genes carry a subscript “GI” [genomic island] if an ortholog of the same gene is present in genomes of E. coli.) LHR-encoded heat shock proteins sHSP20, ClpKGI, and sHSPGI are not sufficient for the heat resistance phenotype; YfdX1, YfdX2, and HdeD are necessary to complement the LHR heat shock proteins and to impart a high level of resistance. Deletion of trx GI, kefB, and psiE GI from plasmid-encoded copies of the LHR did not significantly affect heat resistance. The effect of the growth phase and the NaCl concentration on expression from the putative LHR promoter p2 was determined by quantitative reverse transcription-PCR and by a plasmid-encoded p2:GFP promoter fusion. The expression levels of exponential- and stationary-phase E. coli cells were not significantly different, but the addition of 1% NaCl significantly increased LHR expression. Remarkably, LHR expression in E. coli was dependent on a chromosomal copy of evgA. In conclusion, this study improved our understanding of the genes required for exceptional heat resistance in E. coli and factors that increase their expression in food. IMPORTANCE The locus of heat resistance (LHR) is a genomic island conferring exceptional heat resistance to several foodborne pathogens. The exceptional level of heat resistance provided by the LHR questions the control of pathogens by current food processing and preparation techniques. The function of LHR-comprising genes and their regulation, however, remain largely unknown. This study defines a core complement of LHR-encoded proteins that are necessary for heat resistance and demonstrates that regulation of the LHR in E. coli requires a chromosomal copy of the gene encoding EvgA. This study provides insight into the function of a transmissible genomic island that allows otherwise heat-sensitive enteric bacteria, including pathogens, to lead a thermoduric lifestyle and thus contributes to the detection and control of heat-resistant enteric bacteria in food.

scholarly journals The Cpx Stress Response System Potentiates the Fitness and Virulence of Uropathogenic Escherichia coli

2013 ◽  
Vol 81 (5) ◽  
pp. 1450-1459 ◽  
Author(s):  
Irina Debnath ◽  
J. Paul Norton ◽  
Amelia E. Barber ◽  
Elizabeth M. Ott ◽  
Bijaya K. Dhakal ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Stress Response ◽  
Aminoglycoside Antibiotic ◽  
Deletion Mutants ◽  
Content Type ◽  
Response System ◽  
Stress Response System ◽  
Tract Infections ◽  
Auxiliary Factor

ABSTRACTStrains of uropathogenicEscherichia coli(UPEC) are the primary cause of urinary tract infections, representing one of the most widespread and successful groups of pathogens on the planet. To colonize and persist within the urinary tract, UPEC must be able to sense and respond appropriately to environmental stresses, many of which can compromise the bacterial envelope. The Cpx two-component envelope stress response system is comprised of the inner membrane histidine kinase CpxA, the cytosolic response regulator CpxR, and the periplasmic auxiliary factor CpxP. Here, by using deletion mutants along with mouse and zebrafish infection models, we show that the Cpx system is critical to the fitness and virulence of two reference UPEC strains, the cystitis isolate UTI89 and the urosepsis isolate CFT073. Specifically, deletion of thecpxRAoperon impaired the ability of UTI89 to colonize the murine bladder and greatly reduced the virulence of CFT073 during both systemic and localized infections within zebrafish embryos. These defects coincided with diminished host cell invasion by UTI89 and increased sensitivity of both strains to complement-mediated killing and the aminoglycoside antibiotic amikacin. Results obtained with thecpxPdeletion mutants were more complicated, indicating variable strain-dependent and niche-specific requirements for this well-conserved auxiliary factor.

scholarly journals Proteomic and Transcriptomic Analysis of Microviridae φX174 Infection Reveals Broad Upregulation of Host Escherichia coli Membrane Damage and Heat Shock Responses

mSystems ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Bradley W. Wright ◽  
Dominic Y. Logel ◽  
Mehdi Mirzai ◽  
Dana Pascovici ◽  
Mark P. Molloy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Host Response ◽  
Membrane Damage ◽  
Host Responses ◽  
Human Gut ◽  
Content Type ◽  
E Coli ◽  
Shock Proteins

ABSTRACT Measuring host-bacteriophage dynamics is an important approach to understanding bacterial survival functions and responses to infection. The model Microviridae bacteriophage φX174 is endemic to the human gut and has been studied for over 70 years, but the host response to infection has never been investigated in detail. To address this gap in our understanding of this important interaction within our microbiome, we have measured host Escherichia coli C proteomic and transcriptomic response to φX174 infection. We used mass spectrometry and RNA sequencing (RNA-seq) to identify and quantify all 11 φX174 proteins and over 1,700 E. coli proteins, enabling us to comprehensively map host pathways involved in φX174 infection. Most notably, we see significant host responses centered on membrane damage and remodeling, cellular chaperone and translocon activity, and lipoprotein processing, which we speculate is due to the peptidoglycan-disruptive effects of the φX174 lysis protein E on MraY activity. We also observe the massive upregulation of small heat shock proteins IbpA/B, along with other heat shock pathway chaperones, and speculate on how the specific characteristics of holdase protein activity may be beneficial for viral infections. Together, this study enables us to begin to understand the proteomic and transcriptomic host responses of E. coli to Microviridae infections and contributes insights to the activities of this important model host-phage interaction. IMPORTANCE A major part of the healthy human gut microbiome is the Microviridae bacteriophage, exemplified by the model φX174 phage, and their E. coli hosts. Although much has been learned from studying φX174 over the last half-century, until this work, the E. coli host response to infection has never been investigated in detail. We reveal the proteomic and transcriptomic pathways differentially regulated during the φX174 infection cycle and uncover the details of a coordinated cellular response to membrane damage that results in increased lipoprotein processing and membrane trafficking, likely due to the phage antibiotic-like lysis protein. We also reveal that small heat shock proteins IbpA/B are massively upregulated during infection and that these holdase chaperones are highly conserved across the domains of life, indicating that reliance on them is likely widespread across viruses.

Expression of heat shock proteins in medulloblastoma

2013 ◽  
Vol 12 (5) ◽  
pp. 452-457 ◽  
Author(s):  
George A. Alexiou ◽  
George Vartholomatos ◽  
Kalliopi Stefanaki ◽  
Amalia Patereli ◽  
Lefkothea Dova ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Significant Positive Correlation ◽  
Large Cell ◽  
Hsp70 Expression ◽  
Ki 67 ◽  
Positive Correlation ◽  
Wide Range ◽  
Shock Proteins ◽  
Multiplex Bead

Object Medulloblastoma (MB) is the most common malignant brain tumor in children. Heat shock proteins (HSPs) comprise a superfamily of proteins that serve as molecular chaperones and are overexpressed in a wide range of human cancers. The purpose of the present study was to investigate the expression of HSP27 (pSer82), HSP27 (pSer15), HSP40, HSP60, HSP70, HSP90-α, Akt, and phospho-Akt by multiplex bead array assay of MBs. The results of HSP and Akt expression were correlated with MB subtype; immunohistochemical expression of Ki-67 index, bcl-2, and p53; and patients' prognosis. Methods The authors retrospectively evaluated 25 children with MB who underwent surgery. Immunohistochemical analysis of Ki-67, p53, and bcl-2 expression was performed in all cases. By using multiplex bead array assay, a simultaneous detection of HSP27 (pSer82), HSP27 (pSer15), HSP40, HSP60, HSP70, HSP90-α, Akt, and phospho-Akt was performed. Results Medulloblastoma with extensive nodularity had significantly lower HSP27 (pSer15) expression (p = 0.039) but significantly higher HSP60 expression (p = 0.021) than classic MB. Large-cell MB had significantly higher HSP70 expression (p = 0.028) than classic MB. No significant difference was found between HSP27 (pSer82), HSP40, HSP90-α, Akt, or phospho-Akt expression and MB subtype. Large-cell MBs had significantly higher Ki-67 index compared with classic MBs (p = 0.033). When analyzing all MBs, there was a significant negative correlation between HSP27 (pSer15) and Ki-67 index (r = −0.475, p = 0.016); a significant positive correlation between HSP70 expression and Ki-67 index (r = 0.407, p = 0.043); and a significant positive correlation between HSP70 expression and bcl-2 index (r = 0.491, p = 0.023). Patients with large-cell MB had a worse survival than those with classic MB, but the difference did not reach statistical significance (p = 0.076). Conclusions A substantial expression of several HSPs in MB was observed. Given that HSPs represent an attractive strategy for anticancer therapy, further studies, involving larger series of patients, are obviously necessary to clarify the relationship of HSPs with tumor aggressiveness and prognosis.

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