Influence of the ferric uptake regulator (Fur) protein on pathogenicity in Pectobacterium carotovorum subsp brasiliense
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Date
Authors
Tanui, Collins Kipngetich
Shyntum, Divine Yufetar
Priem, Stefan Louis
Theron, Jacques
Moleleki, Lucy N.
Journal Title
Journal ISSN
Volume Title
Publisher
Public Library of Science
Abstract
Iron is an important nutrient for the survival and growth of many organisms. In order to survive,
iron uptake from the environment must be strictly regulated and maintained to avoid
iron toxicity. The ferric uptake regulator protein (Fur) regulates genes involved in iron
homeostasis in many bacteria, including phytopathogens. However, to date, the role played
by Fur in the biology of Pectobacterium carotovorum subsp. brasiliense (Pcb1692), an
important pathogen of potatoes, has not yet been studied. To this end, we used the lambda
recombineering method to generate a fur mutant strain of Pcb1692 and assessed the virulence
and fitness of the mutant strain. The results showed that production of siderophores in
Pcb1692Δfur increased compared to the Pcb1692 wild-type and the complemented strain
Pcb1692Δfur-pfur. However, production of N-acyl homoserine lactone (AHLs), biofilm formation,
exopolysaccharide (EPS) production, virulence on potato tubers and swimming
motility, were all significantly decreased in Pcb1692Δfur compared to the wild-type and complemented
Pcb1692Δfur-pfur strains. The Pcb1692Δfur mutant also demonstrated significant
sensitivity to oxidative stress when exposed to H2O2. Consistent with phenotypic
results, qRT-PCR results demonstrated that Fur down-regulates genes which encode proteins
associated with: iron uptake (HasA-extracellular heme-binding protein and Ferrodoxin-
AED-0004132), stress response (SodC-superoxide dismutase), plant cell wall degrading
enzymes (PrtA and CelV) and motility (FlhC and MotA). We conclude that the ferric uptake
regulator protein (Fur) of Pcb1692 regulates traits that are important to host-pathogens
interactions.
Description
S1 Fig. Schematic representation of how Pcb1692Δfur mutant strain was generated. A)
Using specific set of primers, PCR amplifications of the fur upstream and downstream regions
were generated as indicated in S1A Fig. Kanamycin cassette was amplified from pKD4 plasmid
with primers Kan F and Kan R. B) Primers Fur F and R, were used in a PCR reaction consisting
of, the fur upstream kanamycin and downstream PCR fragment to generate a PCR fusion
product. C) The fusion product was electroporated into electrocompetent Pcb1692 to generate
the Pcb1692Δfur mutant strain (S1D Fig). Both electrocompetent Pcb1692 and Pcb1692Δfur
mutant strain were electroporated with empty pTrc99A.
S2 Fig. PCR amplicons used to generate the fur mutant. Lane 1. DNA ladder, 2. fur downstream PCR fragment, 3. kanamycin cassette PCR product, 4. fur upstream PCR fragment, 5. Fusion product consisting of the downstream, kanamycin and upstream fragment. 6. The fragment used for complementation which contains the fur gene and its promoter region. 7. Control.
S3 Fig. Schematic presentation of the putative promoter regions of selected genes in Pcb1692. Based on our qRT-PCR results, some of the genes under the Pcb1692 Fur regulon were aligned to the consensus fur box and the putative fur boxes for each gene is indicated by red boxes.
S2 Fig. PCR amplicons used to generate the fur mutant. Lane 1. DNA ladder, 2. fur downstream PCR fragment, 3. kanamycin cassette PCR product, 4. fur upstream PCR fragment, 5. Fusion product consisting of the downstream, kanamycin and upstream fragment. 6. The fragment used for complementation which contains the fur gene and its promoter region. 7. Control.
S3 Fig. Schematic presentation of the putative promoter regions of selected genes in Pcb1692. Based on our qRT-PCR results, some of the genes under the Pcb1692 Fur regulon were aligned to the consensus fur box and the putative fur boxes for each gene is indicated by red boxes.
Keywords
Iron, Protein, Enzymes, Ferric uptake regulator (Fur), Pathogenicity, Pectobacterium carotovorum subsp. brasiliense
Sustainable Development Goals
Citation
Tanui CK, Shyntum DY, Priem SL, Theron
J, Moleleki LN (2017) Influence of the ferric uptake
regulator (Fur) protein on pathogenicity in
Pectobacterium carotovorum subsp. brasiliense.
PLoS ONE 12(5): e0177647. https://DOI.org/ 10.1371/journal.pone.0177647.