Identification of Methanoculleus spp. as active methanogens during anoxic incubations of swine manure storage tank samples

Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-¹³C] acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of ¹³C into DNA was detectable at in situ acetate concentrations (~7g/liter). Fingerprints of DNA retrieved from heavy fractions of the ¹³C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis

Correction: Functional implications of hexameric assembly of RraA proteins from Vibrio vulnificus.

[This corrects the article DOI: 10.1371/journal.pone.0190064.]

Functional implications of hexameric assembly of RraA proteins from Vibrio vulnificus.

RNase E has a pivotal role in the degradation and processing of RNAs in Escherichia coli, and protein inhibitors RraA and RraB control its enzymatic activity. The halophilic pathogenic bacterium Vibrio vulnificus also expresses orthologs of RNase E and RraA-RNase EV, RraAV1, and RraAV2 (herein renamed as VvRNase E, VvRraA1, and VvRraA2). A previous study showed that VvRraA1 actively inhibits the ribonucleolytic activity of VvRNase E by interacting with the C-terminal region of VvRNase E. However, the molecular mechanism underlying the effect of VvRraA1 on the ribonucleolytic activity of VvRNase E has not yet been elucidated. In this study, we report that the oligomer formation of VvRraA proteins affects binding efficiency to VvRNase E as well as inhibitory activity on VvRNase E action. The hexameric structure of VvRraA1 was converted to lower oligomeric forms when the Cys 9 residue was substituted with an Asp residue (VvRraA1-C9D), showing decreased inhibitory activity of VvRraA1 on VvRNase E in vivo. These results indicated that the intermolecular disulfide linkage contributed critically to the hexamerization of VvRraA1 for its proper function. On the contrary, the VvRraA2 that existed in a trimeric state did not bind to or inhibit VvRNase E. An in vitro cleavage assay further showed the reduced inhibitory effect of VvRraA-C9D on VvRNase E activity compared to wild-type VvRraA1. These findings provide insight into how VvRraA proteins can regulate VvRNase E action on its substrate RNA in V. vulnificus. In addition, based on structural and functional comparison of RraA homologs, we suggest that hexameric assembly of RraA homologs may well be required for their action on RNase E-like proteins

Inhibition of VvRraA1 and VvRraA1-C9D on the cleavage of p-BR10+hpT by VvRNase E <i>in vitro</i>.

<p>0.5 pmol of 5’-end-labeled p-BR10+hpT RNA was incubated with 1 pmol of VvRne with varying concentrations of VvRraA1 and VvRraA1-C9D, 50 pmol of VvRraA1, or 50 pmol of BSA in 20 μl of 1 × cleavage buffer at 37°C for 2 h for VvRne, VvRraA1 only, or BSA only controls. Samples were mixed with an equal volume of loading buffer, and then denatured at 65°C for 5 min and loaded onto a 12% polyacrylamide gel containing 8 M urea. The percentage of uncleaved p-BR10+hpT in the gel was quantitated using a phosphorimager and OptiQuant software.</p

List of strains and plasmids used in the present work.

<p>List of strains and plasmids used in the present work.</p

Alignment of amino acid sequences of <i>E</i>. <i>coli</i> RraA and its orthologs in Gram-negative bacteria.

<p>(A) Alignment of amino acid sequence using CLUSTAL W. VvRraA1 and VvRraA2; Amino acid sequences of RraA homologs from <i>E</i>. <i>coli</i> (EcRraA), <i>Mycobacterium tuberculosis</i> (MtRraA), <i>P</i>. <i>aeruginosa</i> (PaRraA), <i>Vibrio cholerae</i> (VcRraA), <i>V</i>. <i>vulnificus</i> (VvRraA1 and VvRraA2) are used. Arrows indicate conserved Cys9 and Cys41 residues of RraA proteins. (B) A molecular model for the C9D mutant of <i>E</i>. <i>coli</i> RraA. The model of the mutant protein was built based on the wild-type structure of <i>E</i>. <i>coli</i> RraA (PDB code: 1Q5X). The subunits are displayed in different colors (cyan and yellow). The mutated Asp9 is positioned in the hydrophobic pocket lined with residues in gold at the interface between the two neighboring subunits, which would destabilize the oligomeric forms of the protein (left lower box). The near region of Cys9 structure is shown in the left upper box.</p

Oligomerization state of EcRraA protein.

<p>Size exclusion chromatography with MALS of EcRraA was performed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190064#pone.0190064.g002" target="_blank">Fig 2</a>. <i>a</i>, 87.05 kDa (~hexamer).</p