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The extracellular proteome of two Bifidobacterium strains reveals different adaptation strategies to low iron conditions

ABSTRACT: B. kashiwanohense PV20-2 and B. pseudolongum PV8-2 are strains isolated from breast fed iron deficient Kenyan infants, selected for their high iron sequestration mechanisms and their genome was completely sequenced. Based on their high iron sequestration features we hypothesized that B. kashiwanohense PV20-2 and B. pseudolongum PV8-2, possess iron related genes and excrete iron binding proteins in the culture media under iron limited conditions. Thus, the complete genomes of B. kashiwanohense PV20-2 and B. pseudolongum PV8-2 were compared to other bifidobacterial genomes to identify genes potentially involved in iron metabolism and the coding sequences from the genome were used as a scaffold to identify the extracellular proteome of both strains grown under low iron conditions using a gel-based shotgun proteomic approach.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Bifidobacterium Pseudolongum

TISSUE(S): Primary Cell Line Cell

SUBMITTER: Pamela Vazquez-Gutierrez

LAB HEAD: Prof. Christophe Lacroix

PROVIDER: PXD001452 | Pride | 2015-12-14

REPOSITORIES: Pride

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Project description:Pyruvate oxidase encoded by spxB is a major virulence factor in the human respiratory pathogen Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes large amounts of H2O2 and acetyl phosphate, which can serve as a phosphoryl group donor to response regulators and be converted to ATP. SpxB is the main source of the millimolar concentrations of H2O2 produced and tolerated by pneumococcus, despite its lack of a catalase. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen, similar to those used previously for phase variants, for spontaneous mutations that caused colonies of virulent serotype 2 strain D39 to change from a transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency of 3 x 10-5) were impaired for SpxB function. Modeling suggested that two mutations changed amino acids in SpxB required for FAD cofactor or subunit binding. One mutation deleted a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three independent mutations created the same frameshift near the start of a trans-acting regulatory gene designated as spxR. The SpxR protein contains helix-turn-helix, CBS, and HotDog domains implicated in DNA, adenosine, and CoA compound binding, respectively, consistent with the idea that SpxR positively regulates spxB transcript amount in response to energy and metabolic state rather than oxidative state. Finally, microarray analyses of a null spxB or a spxR mutant revealed the presence of a new oxidative stress response in pneumococcus and unexpectedly demonstrated that SpxR strongly positively regulates the transcript amount of the strH exoglycosidase gene, which like spxB, has been implicated in host colonization. Keywords: genetic modification Bacterial strains were grown exponentially in rich (BHI) media at 37C and an atmosphere of 5% CO2, and were processed as described in the related Sample records. Samples were collected from three independent biological replicates and included one dye swap. Data were normalized using the Lowess (subgrid) method without background subtraction.

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The extracellular proteome of two Bifidobacterium strains reveals different adaptation strategies to low iron conditions

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