Physiological response of bacillus licheniformis NCIMB 8874 to oligosaccharide elicitors

Reffatti, P. 2012. Physiological response of bacillus licheniformis NCIMB 8874 to oligosaccharide elicitors. PhD thesis University of Westminster School of Life Sciences

TitlePhysiological response of bacillus licheniformis NCIMB 8874 to oligosaccharide elicitors
TypePhD thesis
AuthorsReffatti, P.
Abstract

The mechanism by which certain oligosaccharides elicit overproduction of

secondary metabolites in Bacillus licheniformis was investigated. Mannan

oligosaccharides (MO) derived from locust bean gum, and oligoguluronate (OG)

and oligomannuronate (OM) from alginate were used in this study.

The effect of lower (50 - 25 mg L-1) and higher (500 – 1000 mg L-1)

concentrations of MO, OG and OM on bacitracin A production was investigated.

HPLC (high performance liquid chromatography) results demonstrated that the

addition of lower and higher concentrations of these elicitors to cultures of

B. licheniformis did not have an elicitation effect on bacitracin A production,

compared to the optimal reported elicitor concentration (100 – 300 mg L-1).

HPAEC (high performance anion exchange chromatography) and TLC (thin layer

chromatography) results revealed that mannobiose, mannose and mannohexaose

are the main components of MO. The addition of these saccharides, separately, to B. licheniformis cultures, showed that mannobiose produced the highest and

sustained increase in bacitracin A concentration whereas in mannose and

mannohexaose treated cultures the effects were minimal. Similar levels of

bacitracin A were obtained in MO (825 mg L-1) and mannobiose (800 mg L-1)

treated cultures. Investigation of the fate of MO during the B. licheniformis shake flask and bioreactor batch growth showed that MO is degraded to mannose by the

enzyme ß-mannanase.

MO (300 mg L-1) supplemented chemostat cultures of B. licheniformis did not

result in a significant change in bacitracin A concentration and ß-mannanase

activity compared to the control fermentation. However, when the chemostat

culture was changed to batch, increases were observed both in bacitracin A

concentration and ß-mannanase activity.

Incubation of B. licheniformis cells with oligosaccharide elicitors in microwell plates resulted in changes in Ca2+ influx. The highest influx was 9.2 % greater in samples incubated with MO and OG compared to the control. Addition of

ionomycin (positive control) and verapamil (Ca+2 channel blocker) to the cultures of B. licheniformis resulted in 15 % increase and 74 % decrease in bacitracin A

levels, respectively, as compared to the control culture.

Changes were profiled in protein expression in B. licheniformis supplemented

with OG and MO elicitors in short and long term elicitation, using

2-Dimentional gel electrophoresis. Differentially expressed proteins were

identified by LC-MS/MS. The proteins affected by short-term and long-term

elicitation were mainly involved in energy generation, amino acid metabolism and stress responses. The addition of OG and MO to the cultures also altered the

phosphorylation state of twelve proteins involved in energy generation, stress

response and amino acid metabolism. Biochemical assays revealed changes in

reactive oxygen species levels as well as antioxidant enzymes, superoxide

dismutase and catalase in B. licheniformis cultures supplemented with elicitors. The novel findings of this work have advanced understanding of the mechanism

of action of elicitors in B. licheniformis cultures.

YearJul 2012
FilePatricia_REFFATTI.pdf
Publication dates
CompletedJul 2012

Related outputs

Evidence for the involvement of intracellular Ca 2+ ions in the elicitation mechanism of Bacillus Licheniformis
Reffatti, P., Roy, I., Odell, M. and Keshavarz, T. 2013. Evidence for the involvement of intracellular Ca 2+ ions in the elicitation mechanism of Bacillus Licheniformis. Journal of Molecular Microbiology and Biotechnology. OnlineFirst.

Permalink - https://westminsterresearch.westminster.ac.uk/item/8z360/physiological-response-of-bacillus-licheniformis-ncimb-8874-to-oligosaccharide-elicitors


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