The inside scoop: Comparative genomics of two intranuclear bacteria, "Candidatus Berkiella cookevillensis" and "Candidatus Berkiella aquae"

Abstract

"Candidatus Berkiella cookevillensis" (strain CC99) and "Candidatus Berkiella aquae" (strain HT99), belonging to the Coxiellaceae family, are gram-negative bacteria isolated from amoebae in biofilms present in human-constructed water systems. Both bacteria are obligately intracellular, requiring host cells for growth and replication. The intracellular bacteria-containing vacuoles of both bacteria closely associate with or enter the nuclei of their host cells. In this study, we analyzed the genome sequences of CC99 and HT99 to better understand their biology and intracellular lifestyles. The CC99 genome has a size of 2.9Mb (37.9% GC) and contains 2,651 protein-encoding genes (PEGs) while the HT99 genome has a size of 3.6Mb (39.4% GC) and contains 3,238 PEGs. Both bacteria encode high proportions of hypothetical proteins (CC99: 46.5%; HT99: 51.3%). The central metabolic pathways of both bacteria appear largely intact. Genes for enzymes involved in the glycolytic pathway, the non-oxidative branch of the phosphate pathway, the tricarboxylic acid pathway, and the respiratory chain were present. Both bacteria, however, are missing genes for the synthesis of several amino acids, suggesting reliance on their host for amino acids and intermediates. Genes for type I and type IV (dot/icm) secretion systems as well as type IV pili were identified in both bacteria. Moreover, both bacteria contain genes encoding large numbers of putative effector proteins, including several with eukaryotic-like domains such as, ankyrin repeats, tetratricopeptide repeats, and leucine-rich repeats, characteristic of other intracellular bacteria.

Fig 1. Protozoa infected with “Ca. B. cookevillensis” (CC99) or “Ca. B. aquae” (HT99).

Electron micrograph (13,000x magnification) of intracellular CC99 (arrow) exhibiting coccoid morphology following exposure of the BCV by the tape ripping technique (A). Micrograph of adherent HT99 (arrow) on the surface of A. polyphaga exhibiting coccobacillus morphology (20,000x magnification) (B). Giemsa staining of D. discoideum (strain AX2) infected with CC99 (C) and HT99 (D) showing bacteria (arrow; dark purple) associated with nuclei (pink).

Fig 2. Structural features of “Ca. B. cookevillensis” (CC99) and “Ca. B. aquae” (HT99) genomes.

Genome tracks show (from inner to outer) the positive (green) and negative (purple) GC skew [(C-G)/(C+G)], G+C content (black) and the coding DNA sequences (blue) located in the reverse and forward and strands. Putative origin of replication is located at the top. CGview (http://wishart.biology.ualberta.ca/cgview/) was used to construct the genome map.

Fig 3. Genome inferred central carbohydrate metabolic pathway of “Ca. B. cookevillensis” (CC99) and “Ca. B. aquae” (HT99) with comparisons to C. burnetii and L. pneumophila.

Genes encoding enzymes involved in the EMP of glycolysis (except for glucokinase) and non-oxidative branch of the PPP were identified in both CC99 and HT99 (indicated by blue and green arrows, respectively). Genes for enzymes involved in the oxidative branch of PPP pathway and the ED pathway were absent. Genes encoding enzymes involved in the TCA cycle were present in both bacteria. Genes encoding enzymes for these pathways in C. burnetii (yellow arrows) and L. pneumophila (pink arrows) are included for comparison.

Fig 4. Amino acid biosynthesis pathways of “Ca. B. cookevillensis” (CC99) and “Ca. B. aquae” (HT99).

Genome inferred amino acid pathways appear to be reduced in both bacteria. Only 11 of the 20 amino acid pathways could be asserted for CC99 while only 8 could be asserted for HT99.

Fig 5. Genes involved in the synthesis of flagella in bacteria.

Gene homologs encoding flagellar proteins identified in “Ca. B. cookevillensis” (CC99) and “Ca. B. aquae” (HT99). Genes encoding motor/switch, basal body, hook, filament, filament cap proteins were identified.

Fig 6. Dot/icm T4SS in “Ca. B. cookevillensis” (CC99) and “Ca. B. aquae” (HT99) with comparisons to C. burnetii and L. pneumophila.

(A) Presumed location and topological relationships of dot/icm proteins. Figure adapted from [77]. (B) Genomic organization of the genes encoding dot/icm proteins in CC99 and HT99. Genes dotC, dotD, dotF/icmG, dotG/icmE and dotH/icmK encode the core proteins of the complex. Genes icmS and icmW encode components involved substrate recognition and secretion. Both bacteria are missing the gene encoding LvgA, a protein identified as a potential fifth chaperone to IcmS. Genes dotA, dotE, dotI, dotP and icmV which encode the inner membrane proteins are also present in both bacteria. Genes dotI and dotJ (absent in both bacteria) encode for an integral inner membrane protein. Genes dotL, dotM and dotN encode membrane proteins components involved in recruitment of effector proteins. Genes dotB and dotO encode cytoplasmic ATPase complex proteins. Genes dotV and dotJ (encoding inner membrane proteins); icmQ (encoding a pore-forming cytoplasmic protein) and icmR (encoding a chaperone for IcmQ) are absent in both bacteria.