Abstract
ABSTRACTThe formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. The analyses revealed that 97 genes and 56 proteins were significantly changed upon VBNC state entry. Genes and proteins related to membrane transport, central metabolisms, DNA replication, and cell division were mainly downregulated in the VBNC cells. This caused low metabolic activity concurrently with a division arrest in cells, which may be related to VBNC state formation. Cell division repression and outer membrane overexpression were confirmed to be involved in VBNC state formation by homologous expression of z2046 coding for transcriptional repressor and ompF encoding outer membrane protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a low level of ATP. Combating the low energy supply, ATP production in the VBNC cells was compensated by the degradation of l-serine and l-threonine, the increased AMP generation, and the enhanced electron transfer. Furthermore, tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO2 stresses was enhanced by promoting the production of ammonia and NADPH and by reducing CO2 production during VBNC state formation. Most genes and proteins related to pathogenicity were downregulated in the VBNC cells. This would decrease the cell pathogenicity, which was confirmed by adhesion assays. In conclusion, the decreased metabolic activity, repressed cell division, and enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state formation.
Highlights
The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation proteomic methods
RNA-Seq transcriptomics combined with the use of the isobaric tag for relative and absolute quantitation (iTRAQ) proteomic method was performed to analyze differentially expressed genes and proteins in VBNC E. coli O157:H7 induced by HPCD treatment and to provide new insights into VBNC state formation
Using transmission electron microscopy (TEM), we found that the nucleoid structure was loosened in the VBNC cells [5], which corresponds to the fact of SeqA overproduction upon VBNC state entry
Summary
The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. We used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by HPCD treatment. Using RNA sequencing (RNA-Seq), Su et al [13] pointed out that the presence of the up- and downregulated genes involved in ATP accumulation, protein modification, and membrane proteins would benefit the survival of VBNC Rhodococcus biphenylivorans under oligotrophic and low-temperature conditions Those studies mainly focused on the metabolic features of the VBNC state rather than on VBNC state formation; they lacked functional verification for transcriptomic or proteomic analyses. RNA-Seq transcriptomics combined with the use of the iTRAQ proteomic method was performed to analyze differentially expressed genes and proteins in VBNC E. coli O157:H7 induced by HPCD treatment and to provide new insights into VBNC state formation. A putative mechanism of VBNC state formation induced by HPCD is proposed in this report
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