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

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.

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Proteomic and transcriptomic analysis of Microviridae φXI74 infection reveals broad up-regulation of host membrane damage and heat shock responses

10.1101/2020.10.26.355149 ◽

2020 ◽

Author(s):

Bradley W Wright ◽

Dominic Y Logel ◽

Mehdi Mirzai ◽

Dana Pascovici ◽

Mark P Molloy ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Proteins ◽

Host Response ◽

Membrane Damage ◽

Small Heat Shock Proteins ◽

Host Responses ◽

Human Gut ◽

Transcriptomic Response ◽

E Coli ◽

Shock Proteins

ABSTRACTMeasuring 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 seventy 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-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 phage.IMPORTANCEA major part of the healthy human gut microbiome are the Microviridae bacteriophage, exemplified by the model φX174 phage. 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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01400-17 ◽

2017 ◽

Vol 83 (20) ◽

Cited By ~ 10

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.

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Heat Shock Protein-Mediated Resistance to High Hydrostatic Pressure in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.2660-2666.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 2660-2666 ◽

Cited By ~ 97

Author(s):

Abram Aertsen ◽

Kristof Vanoirbeek ◽

Philipp De Spiegeleer ◽

Jan Sermon ◽

Kristel Hauben ◽

...

Keyword(s):

Escherichia Coli ◽

High Pressure ◽

Hydrostatic Pressure ◽

Heat Shock ◽

Heat Shock Proteins ◽

High Hydrostatic Pressure ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

E Coli ◽

Shock Proteins

ABSTRACT A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i) the expression of rpoH, encoding the heat shock-specific sigma factor σ32, was also induced by high pressure; (ii) heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii) basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.

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Thermoprotective properties of small heat shock proteins from rice, tomato and Synechocystis sp. PCC6803 overexpressed in, and isolated from, Escherichia coli

Functional Plant Biology ◽

10.1071/pp01107 ◽

2001 ◽

Vol 28 (12) ◽

pp. 1219

Author(s):

Carl S. Pike ◽

Joanne Grieve ◽

Murray R. Badger ◽

G. Dean Price

Keyword(s):

Escherichia Coli ◽

Heat Shock ◽

Photosystem Ii ◽

Heat Shock Proteins ◽

Thermal Aggregation ◽

E Coli ◽

Synechococcus Sp ◽

Shock Proteins ◽

Synechocystis Sp

The present study forms part of a program investigating the role of small heat shock proteins (sHSPs) in the acquired and transgenic thermotolerance of the cyanobacterium Synechococcus PCC7942. The genes for three minimally related sHSPs, OsHSP from Oryza sativacytoplasm, tom111 from Lycopersicon esculentumchloroplasts, and 6803 HSP from Synechocystis sp. PCC6803, were cloned into the Escherichia coli vector pTrcHisA, so as to produce an N-terminal polyhistidine tag. The genes were transformed into E. coli and overexpressed. The tagged HSPs were purified (not completely in the case of tom111) by immobilised metal affinity chromatography. The native proteins exhibited a high degree of oligomerisation when analysed by size-exclusion chromatography. All three proteins were able to protect malate dehydrogenase (MDH) from in vitro thermal aggregation. They could also protect several soluble proteins in E. coli extracts from thermal aggregation in vitro, as well as protecting phycocyanin in extracts from Synechococcus sp. PCC7942. None of the proteins were able to protect photosystem II (measured as ΦPSII, the effective quantum fluorescence yield of PSII) of thylakoids isolated from Synechococcus sp. PCC7942 from heat damage in vitro, although in vivo, after acclimation, photosystem II did exhibit acquired thermotolerance.

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Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance

mBio ◽

10.1128/mbio.00308-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 57

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.

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Roles of the Escherichia coli Small Heat Shock Proteins IbpA and IbpB in Thermal Stress Management: Comparison with ClpA, ClpB, and HtpG In Vivo

Journal of Bacteriology ◽

10.1128/jb.180.19.5165-5172.1998 ◽

1998 ◽

Vol 180 (19) ◽

pp. 5165-5172 ◽

Cited By ~ 130

Author(s):

Jeffrey G. Thomas ◽

François Baneyx

Keyword(s):

Escherichia Coli ◽

Heat Shock ◽

Heat Shock Proteins ◽

High Temperatures ◽

Small Heat Shock Proteins ◽

Host Protein ◽

E Coli ◽

Growth Defects ◽

Shock Proteins

ABSTRACT We have constructed an Escherichia coli strain lacking the small heat shock proteins IbpA and IbpB and compared its growth and viability at high temperatures to those of isogenic cells containing null mutations in the clpA, clpB, orhtpG gene. All mutants exhibited growth defects at 46°C, but not at lower temperatures. However, the clpA,htpG, and ibp null mutations did not reduce cell viability at 50°C. When cultures were allowed to recover from transient exposure to 50°C, all mutations except Δibpled to suboptimal growth as the recovery temperature was raised. Deletion of the heat shock genes clpB and htpGresulted in growth defects at 42°C when combined with thednaK756 or groES30 alleles, while the Δibp mutation had a detrimental effect only on the growth of dnaK756 mutants. Neither the overexpression of these heat shock proteins nor that of ClpA could restore the growth ofdnaK756 or groES30 cells at high temperatures. Whereas increased levels of host protein aggregation were observed indnaK756 and groES30 mutants at 46°C compared to wild-type cells, none of the null mutations had a similar effect. These results show that the highly conserved E. coli small heat shock proteins are dispensable and that their deletion results in only modest effects on growth and viability at high temperatures. Our data also suggest that ClpB, HtpG, and IbpA and -B cooperate with the major E. coli chaperone systems in vivo.

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