Identification of Proteins Associated with Polyhydroxybutyrate Granules from <i>Herbaspirillum seropedicae</i> SmR1 - Old Partners, New Players

<div><p><i>Herbaspirillum seropedicae</i> is a diazotrophic ß-Proteobacterium found associated with important agricultural crops. This bacterium produces polyhydroxybutyrate (PHB), an aliphatic polyester, as a carbon storage and/or source of reducing equivalents. The PHB polymer is stored as intracellular insoluble granules coated mainly with proteins, some of which are directly involved in PHB synthesis, degradation and granule biogenesis. In this work, we have extracted the PHB granules from <i>H. seropedicae</i> and identified their associated-proteins by mass spectrometry. This analysis allowed us to identify the main phasin (PhaP1) coating the PHB granule as well as the PHB synthase (PhbC1) responsible for its synthesis. A <i>phbC1</i> mutant is impaired in PHB synthesis, confirming its role in <i>H. seropedicae.</i> On the other hand, a <i>phaP1</i> mutant produces PHB granules but coated mainly with the secondary phasin (PhaP2). Furthermore, some novel proteins not previously described to be involved with PHB metabolism were also identified, bringing new possibilities to PHB function in <i>H. seropedicae</i>.</p></div

Ribbon diagram of the monomeric crystal structure of HsRecA protein.

<p>Regions that could be modeled were indicated by the last residue-number. HsRecA protein is composed of N-terminal domain (NTD), a central core ATPase domain and a large C-terminal domain (CTD). The core ATPase domain contains one Ca²⁺ ion (magenta sphere), coordinated by Asn119 and Asp120 and the ATPase activity site is partially occupied by ATP and ADP. Figure was prepared using the STRIDE program for secondary structure assignment [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159871#pone.0159871.ref055" target="_blank">55</a>], and visualized using PyMOL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159871#pone.0159871.ref056" target="_blank">56</a>].</p

ATPase profile from EcRecA and HsRecA in the presence and absence of SSB protein.

<p>(A) Reaction 1: contained 5 μM nt M13mp18 cssDNA and 3 μM HsRecA or EcRecA, were previously incubated per 20 min at 37°C, following, 3 μM ATP and 0.5 μM SSB. Reaction 2: reaction 1 without SSB protein addition. Time 0 min indicates the addition of ATP and SSB. (B) The reaction contained 5 μM nt M13mp18 cssDNA, 3 μM ATP and 0.35 μM SSB, were previously incubated per 10 min at 37°C, following addition of 3 μM HsRecA or EcRecA (time 0 min).</p

PHB production of <i>Herbaspirillum seropedicae</i> strains SmR1 (wild-type) and Δ<i>phbC1</i>.

<p>The quantity of PHB was determined as described in Material and Methods during different time of cell growth. Numbers in parenthesis indicate the OD₆₀₀ of the cell culture at the indicated time of growth. Data represent the average of two biological replicates.</p

Expression level of genes related to phasins in <i>Herbaspirillum seropedicae</i>.

a<p>the gene identification is the same as deposited in the Genbank (accession number CP002039.1).</p><p>The expression value corresponds to the average of RPKM of two biological replicates. RPKM is defined as RPKM = total gene reads per mapped reads (millions) × gene length (kb).</p

Electrophoretic pattern of PHB granule-associated proteins from <i>Herbaspirillum seropedicae</i> SmR1 and Δ<i>phaP1</i> strains in 10% SDS-PAGE.

<p>Lanes: MW – molecular weight markers; lane 1: granule-associated proteins after granule purification from the wild type strain SmR1; lane 2: granule-associated proteins after granule purification from the Δ<i>phaP1</i> mutant strain. Phasins are indicated on the right. PhaP1was identified in lane 1 whereas PhaP2 and Hsero_2402 were identified in lane 2. Proteins were Coomassie blue R-250 stained.</p

Structural and Functional Studies of <i>H</i>. <i>seropedicae</i> RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament - Fig 5

<p><b>Omit maps (mF</b>_<b>o</b><b>-DF</b>_<b>c</b><b>) at the ATP binding site:</b> (A) Refinement with ATP at 100% occupancy; the negative electron density over the γ-phosphate indicates that it should not be at this full occupancy. (B) Refinement with ATP at 39% occupancy; the positive electron density over the corresponding ADP moiety indicates that it should be at full occupancy. The omit maps are contoured at +3 (green) and -3 (red) σ levels.</p

DNA strand exchange promoted by the wild-type HsRecA and EcRecA proteins.

<p>(A) The three DNA strand exchange containing 10 <b>μ</b>M nt M13mp18 cssDNA and 3.5 <b>μ</b>M HsRecA or EcRecA, were previously incubated per 20 min at 37°C, and then 3 <b>μ</b>M ATP and 1 <b>μ</b>M SSB were added and incubated for an additional 10 min. The minutes shown represents the time of reaction after addition of 20 <b>μ</b>M nt M13mp18 ldsDNA. (B) The percentage of duplex substrate converted into the nicked circular duplex (NC product) is plotted against the time.</p

Table_1.XLSX

<p>The ability of bacteria to produce polyhydroxyalkanoates such as poly(3-hydroxybutyrate) (PHB) enables provision of a carbon storage molecule that can be mobilized under demanding physiological conditions. However, the precise function of PHB in cellular metabolism has not been clearly defined. In order to determine the impact of PHB production on global physiology, we have characterized the properties of a ΔphaC1 mutant strain of the diazotrophic bacterium Herbaspirillum seropedicae. The absence of PHB in the mutant strain not only perturbs redox balance and increases oxidative stress, but also influences the activity of the redox-sensing Fnr transcription regulators, resulting in significant changes in expression of the cytochrome c-branch of the electron transport chain. The synthesis of PHB is itself dependent on the Fnr1 and Fnr3 proteins resulting in a cyclic dependency that couples synthesis of PHB with redox regulation. Transcriptional profiling of the ΔphaC1 mutant reveals that the loss of PHB synthesis affects the expression of many genes, including approximately 30% of the Fnr regulon.</p

Table_2.XLSX

<p>The ability of bacteria to produce polyhydroxyalkanoates such as poly(3-hydroxybutyrate) (PHB) enables provision of a carbon storage molecule that can be mobilized under demanding physiological conditions. However, the precise function of PHB in cellular metabolism has not been clearly defined. In order to determine the impact of PHB production on global physiology, we have characterized the properties of a ΔphaC1 mutant strain of the diazotrophic bacterium Herbaspirillum seropedicae. The absence of PHB in the mutant strain not only perturbs redox balance and increases oxidative stress, but also influences the activity of the redox-sensing Fnr transcription regulators, resulting in significant changes in expression of the cytochrome c-branch of the electron transport chain. The synthesis of PHB is itself dependent on the Fnr1 and Fnr3 proteins resulting in a cyclic dependency that couples synthesis of PHB with redox regulation. Transcriptional profiling of the ΔphaC1 mutant reveals that the loss of PHB synthesis affects the expression of many genes, including approximately 30% of the Fnr regulon.</p