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Pheno- And Genotyping of Hopanoid Production in Acidobacteria

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Pheno- And Genotyping of Hopanoid Production in Acidobacteria

Jaap S Sinninghe Damsté et al. Front Microbiol.

Abstract

Hopanoids are pentacyclic triterpenoid lipids synthesized by different bacterial groups. Methylated hopanoids were believed to be exclusively synthesized by cyanobacteria and aerobic methanotrophs until the genes encoding for the methylation at the C-2 and C-3 position (hpnP and hpnR) were found to be widespread in the bacterial domain, invalidating their use as specific biomarkers. These genes have been detected in the genome of the Acidobacterium "Ca. Koribacter versatilis," but our knowledge of the synthesis of hopanoids and the presence of genes of their biosynthetic pathway in other member of the Acidobacteria is limited. We analyzed 38 different strains of seven Acidobacteria subdivisions (SDs 1, 3, 4, 6, 8, 10, and 23) for the presence of C30 hopenes and C30+ bacteriohopane polyols (BHPs) using the Rohmer reaction. BHPs and/or C30 hopenes were detected in all strains of SD1 and SD3 but not in SD4 (excepting Chloracidobacterium thermophilum), 6, 8, 10, and 23. This is in good agreement with the presence of genes required for hopanoid biosynthesis in the 31 available whole genomes of cultivated Acidobacteria. All genomes encode the enzymes involved in the non-mevalonate pathway ultimately leading to farnesyl diphosphate but only SD1 and 3 Acidobacteria and C. thermophilum encode all three enzymes required for the synthesis of squalene, its cyclization (shc), and addition and modification of the extended side chain (hpnG, hpnH, hpnI, hpnJ, hpnO). In almost all strains, only tetrafunctionalized BHPs were detected; three strains contained variable relative abundances (up to 45%) of pentafunctionalized BHPs. Only "Ca. K. versatilis" contained methylated hopanoids (i.e., 2,3-dimethyl bishomohopanol), although in low (<10%) amounts. These genes are not present in any other Acidobacterium, consistent with the absence of methylated BHPs in the other examined strains. These data are in agreement with the scattered occurrence of methylated BHPs in other bacterial phyla such as the Alpha-, Beta-, and Gammaproteobacteria and the Cyanobacteria, limiting their biomarker potential. Metagenomes of Acidobacteria were also examined for the presence of genes required for hopanoid biosynthesis. The complete pathway for BHP biosynthesis was evident in SD2 Acidobacteria and a group phylogenetically related to SD1 and SD3, in line with the limited occurrence of BHPs in acidobacterial cultures.

Keywords: Acidobacteria; Hopanoids; genomes; lipid analysis; lipid biosynthesis; metagenomes; methylation.

Figures

Figure 1
Figure 1
Phylogenetic tree of the nearly complete 16S rRNA gene sequences of the Acidobacteria discussed in the text. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. Scale bar indicates 2% sequence divergence. Names in red bold indicate strains in which presence of BHP has been assessed in cultures; names in black bold indicate strains in which the genome has been screened for the presence of the genes discussed in the text, names in blue bold indicate strains in which both the BHP and genome screening has been performed.
Figure 2
Figure 2
GC traces of the products formed by the Rohmer treatment of (A) the total extract and (B) total cell material of Acidobacteriaceae bacterium A2-4c. Released hopanoids are present in the form of hopanols (analyzed as their TMS derivatives). Bacteriohopanetetrol derivatives are transformed by the Rohmer treatment into the C32 hopanol, bacteriohopanepentol derivatives are transformed into the C31 hopanol. The star indicates the internal standard added prior to analysis in a fixed concentration. Since the peak areas of the hopanols relative to the internal standard in (B) is much larger, the hopanol yield is much higher when the Rohmer reaction is applied to total biomass.
Figure 3
Figure 3
Mass spectrum of a putative dimethylated bishomohopanol (as TMS derivative) formed by Rohmer degradation of total cell material of “Ca. Koribacter versatilis” Ellin345. The methylation at position C-2 and C-3 is unprecedented but supported by the indicated mass spectral fragmentation and the presence of both the hpnP and hpnR genes in the genome of “Ca. K. versatilis Ellin345” (Table 2). The indicated stereochemistry of the additional methyl groups is hypothetical.
Figure 4
Figure 4
Biosynthetic scheme of synthesis of BHT derivatives.
Figure 5
Figure 5
Phylogenetic tree of the Shc proteins in the acidobacterial genomes of cultures and environmental genomes. This tree was constructed using the maximum likelihood method with a LG model plus gamma distribution and invariant sites (LG+G+I). The analysis included 750 positions in the final dataset. The scale bar represents number of amino acid substitutions per site. Branch support was calculated with the approximate likelihood ratio test (aLRT) and values ≥50% are indicated on the branches. In general, it reveals the phylogeny that is also apparent from the 16S rRNA gene tree (Figure 1) with distinct clusters for the SD1, SD2, SD3, and SD22 clusters. The only Shc proteins encountered in Acidobacteria SD4 (i.e., in C. thermophilum B and OC1) differ substantially from those of other Acidobacteria and are more closely related to Shc proteins encountered in Cyanobacteria (e.g., Synechoccus sp.). The second Shc protein of the SD1 acidobacterium G. pectinivorans DSM21001 falls in SD3 and is most closely related to the second copy of Shc of “Ca. S. usitatus Ellin 6076.”
Figure 6
Figure 6
Phylogenetic tree of the HpnC, HpnD, and FdfT proteins in the acidobacterial genomes of cultures and environmental genomes. The tree was constructed using the maximum likelihood method with a LG model plus gamma distribution and invariant sites (LG+G+I). The analysis included 509 positions in the final dataset. The scale bar represents the number of amino acid substitutions per site. Branch support was calculated with the approximate likelihood ratio test (aLRT) and values ≥50% are indicated on the branches. The first part (A) of the tree shows the phylogeny of the HpnC and FdfT proteins; zoom in (B) showing the phylogeny of the HpnD proteins. The HpnC tree generally reveals the phylogeny that is also apparent from the 16S rRNA gene tree (Figure 1) with distinct clusters for the SD1, SD2, SD3, SD4, SD6, and SD1/3 acidobacteria. Five species of SD1 acidobacteria also contained an FdtT protein, which is only remotely related to the HpnC and HpnD proteins. These FdtT proteins are closely related to the FdtT proteins of Betaproteobacteria (e.g., Parabulkholderia sartisoli). The HpnC tree generally also reveals the phylogeny that is also apparent from the 16S rRNA gene tree (Figure 1) with distinct clusters for the SD1, SD2, SD3, and SD1/3 acidobacteria. The sequences in the SD2 cluster annotated with an asterisk are annotated as HpnC but in fact represent a fused protein HpnCD (see text and Figure 7). HpnC was considered until the amino acid position 279–339 up to the amino acids RAG/RTG/RVG, while the rest of the protein was cropped and used as a new entry in the alignment and phylogenetic tree. As observed in the Shc protein tree (this figure) the HpnD protein of the SD4 acidobacterium C. thermophilum is only distantly related to the HpnD protein of other acidobacteria. The closest relatives are HpnD proteins of green sulfur bacteria (e.g., Chlorobium limicola and Chlorobaculum tepidum).
Figure 7
Figure 7
Location of BHP biosynthetic genes and gene clusters in the genomes of SD1, SD3, and SD4 Acidobacteria and a number of selected environmental genomes as determined by BLAST protein searches. Species were ordered as indicated by the phylogeny of the shc gene. Stippled lines indicate a distance between the genes. The numbers refer, in combination with the code below the species name, to the locus tag in the annotated genome from the NCBI database. These numbers typically increase by 5 for every next gene. Color codes of genes are: orange, genes involved in the biosynthesis of farnesyl diphosphate; red and dark red (putative), genes involved in the biosynthesis of squalene; blue, genes involved in BHP biosynthesis; yellow, genes involved in the methylation of BHPs; dark green, genes involved in the mevalonate pathway of isoprenoid biosynthesis, light green, genes often of unknown function associated with identified gene clusters. Names of genes refer to Table S1. Three gene clusters were often encountered and these are indicated in different background colors (red, blue, and yellow). When these cluster are indicated with a stippled box, these clusters have been slightly modified by the insertion or deletion of one or a few genes. *There is only a nucleotide sequence corresponding to the open reading frame of hpnC of C. thermophilum B (WP_014101470.1) between amino acids 38–227.

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