Proteomic Phenotyping of Novosphingobium nitrogenifigens Reveals a Robust Capacity for Simultaneous Nitrogen Fixation, Polyhydroxyalkanoate Production, and Resistance to Reactive Oxygen Species

Abstract

<jats:title>ABSTRACT</jats:title><jats:p><jats:named-content content-type="genus-species">Novosphingobium nitrogenifigens</jats:named-content>Y88<jats:sup>T</jats:sup>(Y88) is a free-living, diazotrophic<jats:named-content content-type="genus-species">Alphaproteobacterium</jats:named-content>, capable of producing 80% of its biomass as the biopolymer polyhydroxybutyrate (PHB). We explored the potential utility of this species as a polyhydroxybutyrate production strain, correlating the effects of glucose, nitrogen availability, dissolved oxygen concentration, and extracellular pH with polyhydroxybutyrate production and changes in the Y88 proteomic profile. Using two-dimensional differential in-gel electrophoresis and tandem mass spectrometry, we identified 217 unique proteins from six growth conditions. We observed reproducible, characteristic proteomic signatures for each of the physiological states we examined. We identified proteins that changed in abundance in correlation with either nitrogen fixation, dissolved oxygen concentration, or acidification of the growth medium. The proteins that correlated with nitrogen fixation were identified either as known nitrogen fixation proteins or as novel proteins that we predict play roles in aspects of nitrogen fixation based on their proteomic profiles. In contrast, the proteins involved in central carbon and polyhydroxybutyrate metabolism were constitutively abundant, consistent with the constitutive polyhydroxybutyrate production that we observed in this species. Three proteins with roles in detoxification of reactive oxygen species were identified in this obligate aerobe. The most abundant protein in all experiments was a polyhydroxyalkanoate granule-associated protein, phasin. The full-length isoform of this protein has a long, intrinsically disordered Ala/Pro/Lys-rich N-terminal segment, a feature that appears to be unique to sphingomonad phasins. The data suggest that Y88 has potential as a PHB production strain due to its aerobic tolerance and metabolic orientation toward polyhydroxybutyrate accumulation, even in low-nitrogen growth medium.</jats:p>

Softwood hydrolysate as a carbon source for polyhydroxyalkanoate production

“…In this study the two strains allowed us to compare the impact of batch growth of a strain unaffected by nitrogen deficient conditions (Y88) and a strain impacted by nitrogen deficiency (WP01) in terms of PHA production. Strain Y88 is able to synthesize PHA constitutively and is able to fix atmospheric nitrogen . PHA synthesis in WP01 is induced under conditions of nitrogen limitation; WP01 is not able to fix nitrogen .…”

“…In our previous work total nitrogen of 39 mg L −1 was supplied to Y88 growing on pure glucose, of which 3.6 mg L −1 remain after 20 h of fermentation. It was noted that Y88 switched to nitrogen fixation within the first 20 h of growth before nitrogen was exhausted . In an independent shake flask study using WP01, the optimal nitrogen content for PHA production was established at 100–300 mg L −1 supplied as (NH 4 ) 2 SO 4 which is equivalent to 13.6–40.8 mg L −1 of nitrogen as ammonium …”

“…Strain Y88 was isolated from pulp‐mill wastewater effluent, and WP01 was isolated from soil saturated with extractives released during high pressure steam impregnation of timber with oil at a sawmill site . Both have been characterized taxonomically and their genome sequences determined …”

The Cytosolic Oligosaccharide-Degrading Proteome of Butyrivibrio Proteoclasticus

“…Extracted tryptic peptides were resuspended in 1.5 µL of 10 mg.mL −1 α-cyano-4-hydroxycinnamic acid in 50% acetonitrile: 0.1% trifluoroacetic acid ( v / v ) and analyzed using a Voyager DE Pro MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA) as previously described [10,13,16]. MALDI MS/MS using an ABSCIEX 5800 TOF/TOF mass spectrometer (SCIEX, Framingham, MA, USA) was used for repeat analysis of protein spots that contained fibrolytic enzymes.…”

“…Digestion was stopped by addition of formic acid to 4% ( v / v ) final concentration. LC-MS/MS was carried out as previously described [16] using a Finnigan LTQ™ Linear Ion-trap (Thermo Scientific, Waltham, MA, USA) with a Dionex UltiMate ® 3000 Nano HPLC system (Thermo Scientific, Waltham, MA, USA) containing an Acclaim ® PepMapTM C18 nano-column (75 µm i.d. × 15 cm, 100 Å pore size).…”