Publications

4466

ABSTRACT “ Candidatus Liberibacter asiaticus” is an uncultured alphaproteobacterium that systemically colonizes its insect host both inter- and intracellularly and also causes a severe, crop-destroying disease of citrus called huanglongbing, or citrus “greening.” In planta , “ Ca . Liberibacter asiaticus” is also systemic but phloem limited. “ Ca . Liberibacter asiaticus” strain UF506 carries two predicted prophages, SC1 and SC2. Bacteriophage particles have been observed in experimentally “ Ca . Liberibacter asiaticus”-infected periwinkle but not in any other host. Comparative gene expression analysis of predicted SC1 late genes showed a much higher level of late gene expression, including holin transcripts (SC1_gp110), in “ Ca . Liberibacter asiaticus”-infected periwinkle relative to “ Ca . Liberibacter asiaticus”-infected citrus. To functionally characterize predicted holin and endolysin activity, SC1_gp110 and two predicted endolysins, one within SC1 (SC1_gp035) and another well outside the predicted prophage region (CLIBASIA_04790), were cloned and expressed in Escherichia coli . Both SC1 genes inhibited bacterial growth consistent with holin and endolysin function. The holin (SC1_gp110) promoter region was fused with a uidA reporter on pUFR071, a wide bacterial host range (repW) replicon, and used to transform Liberibacter crescens strain BT-1 by electroporation. BT-1 is the only liberibacter strain cultured to date and was used as a proxy for “ Ca . Liberibacter asiaticus.” pUFR071 was >95% stable without selection in BT-1 for over 20 generations. The reporter construct exhibited strong constitutive glucuronidase (GUS) activity in culture-grown BT-1 cells. However, GUS reporter activity in BT-1 was suppressed in a dose-dependent manner by crude aqueous extracts from psyllids. Taken together with plant expression data, these observations indicate that “ Ca . Liberibacter asiaticus” prophage activation may limit “ Ca . Liberibacter asiaticus” host range and culturability.

Summary In an acetate‐fed anaerobic–aerobic membrane bioreactor with deteriorated enhanced biological phosphorus removal ( EBPR ), D efluviicoccus ‐related tetrad‐forming organisms ( DTFO ) were observed to predominate in the microbial community. Using metagenomics, a partial genome of the predominant DTFO , ‘ C andidatus   D efluviicoccus tetraformis strain TFO 71’, was successfully constructed and characterized. Examining the genome confirmed the presence of genes related to the synthesis and degradation of glycogen and polyhydroxyalkanoate ( PHA ), which function as energy and carbon storage compounds. TFO 71 and ‘ C andidatus   A ccumulibacter phosphatis’ ( CAP ) UW ‐1 and CAP UW ‐2, representative polyphosphate‐accumulating organisms ( PAO ), have PHA metabolism‐related genes with high homology, but TFO 71 has unique genes for PHA synthesis, gene regulation and granule management. We further discovered genes encoding DTFO polyphosphate ( polyP ) synthesis, suggesting that TFO 71 may synthesize polyP under untested conditions. However, TFO 71 may not activate these genes under EBPR conditions because the retrieved genome does not contain all inorganic phosphate transporters that are characteristic of PAOs ( CAP UW ‐1, CAP UW ‐2, M icrolunatus phosphovorus   NM ‐1 and T etrasphaera species). As a first step in characterizing EBPR ‐associated DTFO metabolism, this study identifies important differences between DTFO and PAO that may contribute to EBPR community competition and deterioration.