Figure 1.
Photomicrographs of OHSU_I, OHSU_II and OHSU_III microbes.
A: Photomicrograph of OHSU_II C1 forming microscopic colonies on the surface of a YM agar plate after 2 weeks of incubation at 30°C. The streaked lines on the agar plate could be clearly seen. 40X magnification. B and C: Photomicrographs of OHSU_I C6 and OHSU_II C1 microbes growing at high cell density in the culture broths kept at RT after more than 10 days. The microbes stayed at the bottom of undisturbed culture flasks. Many of the OHSU_I C6 microbes aggregated into clumps and adhered on the plastic flask surface (B). OHSU_II C1 did not aggregate into clumps or adhere on the plastic surface (C). Phase contrast with green filter 400X. D: Photomicrograph of OHSU_III culture. The microbes in the broth appeared to increase in numbers and form loose aggregations (empty arrows) seen at the bottom of the culture flask. However, the microbes could not grow and reach into a high cell density after 3 months of incubation at RT. Fragments of potential blood cells debris were also seen (arrows). Phase contrast 400X
Table 1.
Comparison of growth characteristics, biochemical reactions and G/C contents of Afipia species.
Figure 2.
Thick section photomicrographs and ultrathin electron micrographs of OHSU_I and OHSU_II microbes.
A and B: Thick section photomicrographs of OHSU_I C6 (A) and OHSU_II C1 (B) that were concentrated from cultures using modified SP4 broth. Both sections revealed morphologically similar, slender pointed microbes using longitudinal and cross sectioning. The concentrated microbes were fixed with 2.5% glutaraldehyde, post-fixed with 1% osmium tetroxide and embedded in epoxy resin. The thick sections were stained using 1% toluene blue. 1000X. C and D: Electron photomicrographs of OHSU_I C6 (C) and OHSU_II C1 (D) captured in ultrathin sections. Typical Gram-negative bacteria wall structure (arrows), intracellular ribosomal structures (R), electron-dense bodies and nucleic acid (N) as well as scale bars are indicated for both sections. The ultrathin sections were stained with uranyl acetate and lead citrate. E: Electron micrograph of OHSU_I C6 captured at higher magnification (200,000X). External membrane (black arrow) and internal membrane structures (red arrow) of the microbes are highlighted.
Table 2.
Whole genome sequencing datasheet of new Bradyrhizobiaceae from 3 blood samples.
Figure 3.
Phylogenetic relatedness of OHSU_I, OHSU_II and OHSU_III microbes among different Bradyrhizobiaceae species.
Phylogenetic analysis based on 16S rRNA gene sequences (A) and whole rRNA operon sequences (B) using the neighbor-joining method. GenBank Accession numbers of sequences used in the analyses are shown in parentheses. Scale bar units are estimated branch lengths. Numerals indicate bootstrap percentages over 50 after 500 replications.
Figure 4.
Sequence mapping for draft genomes of A. septicemium grown in OHSU_II and OHSU_I cultures and genome of A. broomeae.
The complete genome of Bradyrhizobiaceae SG-6C was used as the reference to align genomic scaffold supercontigs 1.2, 1.3 and 1.1 of A. broomeae into a working complete genome that was then used as the reference genome for assembling formed contigs from genomic sequencing of the microbes grown in OHSU_I and OHSU_II cultures into draft genomes. The tracks from inside to outside represent GC skews, GC contents, draft genome of OHSU_II microbe, draft genome of OHSU_I microbe and A. broomeae genome. The color blank regions represent sequence differences found between the bacterial genomes. Major regions of difference are seen in the regions of 0.6 Mb and 3 Mb.
Figure 5.
Alignment of whole-genome sequences using draft genomes of OHSU_I isolates (un-cloned, C4 and C6) and OHSU_II isolates (un-cloned, C1 and C2).
Homologous locally collinear blocks among genomes are connected with a line and identified by the same color using progressiveMauve. Blocks that are inverted compared to the OHSU_I (un-cloned) genome are placed under the center line of the genome. Gene rearrangements or inversions among the genomes are indicated. The genome content dissimilarities among these isolates were calculated and are shown in Table 3.
Table 3.
Genome content differences among OHSU_I, OHSU_II isolates of A. septicemium, established Afipia species and Bradyrhizobiaceae SG.
Figure 6.
Sequence mapping for draft genomes of Bradyrhizobium sp. OHSU_III and the genomes of B. elkanii and B. japonicum.
The tracks from inside to outside represent GC skews, GC contents, draft genome of OHSU_III microbe, draft genome B. elkanii and complete genome of B. japonicum.. The color blank regions represent areas of sequence differences found between the bacterial genomes.
Table 4.
Genome content differences among Bradyrhizobium sp. OHSU_III and established Bradyrhizobium species†.