Environmental Microbiology

Summary The X‐bacteria which initiated organismic association with the D strain of Amoeba proteus in 1966 as parasites have changed to obligate endosymbionts on which the host depends for survival. Owing to the difficulty in cultivating the bacteria in vitro , the identity of X‐bacteria has not been determined. As the life cycle of X‐bacteria is similar to that of Legionella spp. in soil amoebae, we applied the polymerase chain reaction method with specific primers aimed at Legionella spp. for the detection and cloning of 16S rRNA gene. The identity and intracellular localization of the endosymbiont were confirmed by the application of a specific fluorescently labelled 16S rRNA‐targeted probe. In addition we cloned RNA polymerase β‐subunit gene ( rpoB ) of X‐bacteria by genomic library tagging. A phylogenetic analysis of the 16S rRNA gene placed the bacterium within a unique monophyletic group containing all other members of the genus Legionella . Phylogeny from rpoB and mip genes further confirmed the taxonomic context of X‐bacteria to be a Legionella sp. In all three phylogenic analyses, X‐bacterium was placed apart from Legionella ‐like amoebal pathogens present in soil amoebae. Thus, we propose the name ‘ Candidatus Legionella jeonii’ sp. nov. for the endosymbiotic X‐bacteria in Amoeba proteus .

Summary ‘ Aegyptianella ranarum ’ (order Rickettsiales), an ultrastructurally defined small, Gram‐negative rod, is known to replicate in the red blood cells of frogs. Heretofore, this bacterium has not been characterized genetically. We cloned and sequenced the 16S rRNA (1310 bp) and gyrB (718 bp) genes of ‘ A. ranarum ’ from a Canadian frog blood specimen. In situ hybridization (with an ‘ A. ranarum ’ 16S rRNA gene polymerase chain reaction product as probe) and electron microscopy confirmed that ‘ A. ranarum ’ forms cytoplasmic inclusions in frog erythrocytes. blast comparisons with GenBank 16S rRNA and gyrB sequences showed that both ‘ A. ranarum ’ genes were most similar (91% and 67% identity) to those of Chryseobacterium meningosepticum , a bacterium in the family Flavobacteriaceae . In contrast, ‘ A. ranarum ’ 16S rRNA shared only 61% identity with Aegyptianella pullorum . Phylogenetic analyses of these genes using phylip supported ‘ A. ranarum ’ as a member of Flavobacteriaceae, but suggested that its cladistic sibling may be Bergeyella zoohelcum or Weeksella virosa , rather than C. meningosepticum. We propose to classify ‘ Aegyptianella ranarum ’ as ‘ Candidatus Hemobacterium ranarum’ in the family Flavobacteriaceae . Our results provide a starting point for studies of related intraerythrocytic bacterial infections in frogs.

Summary Two phylogenetically distinct marine strains producing transparent exopolymers (TEP), designated HTCC2155 T and HTCC2160, were cultivated from Oregon coast seawater by dilution to extinction in a high throughput culturing format. When cultured in low‐nutrient seawater media, these strains copiously produced Alcian Blue‐stainable viscous TEP. Growing cells were attached to each other by the TEP in a three dimensional network. Polymerase chain reaction employing 16S rDNA primers specific for the novel isolates indicated that they are indigenous to the water column of the Atlantic and Pacific oceans. The abundance of the isolates as determined by 16S rRNA dot blots, however, indicated that they are less than 1% of the total bacterial community. In phylogenetic analyses, the strains consistently formed a new phylum‐level lineage within the domain Bacteria , together with members of the candidate phylum VadinBE97, which consists of Victivallis, the first cultured genus in the candidate phylum, and 16S rRNA gene clones from DNA extracted from marine or anaerobic terrestrial habitats. Five putative subgroups were delineated within this phylum‐level lineage, including a marine group and an anaerobic group. The isolates are Gram negative, strictly aerobic, chemoheterotrophic, and facultatively oligotrophic sphere‐shaped bacteria. The DNA G+C content of strain HTCC2155 T was 48.3 mol% and the genome size was 2.9 mb. It is proposed from these observations that the strains be placed into a new genus and a new species named Lentisphaera araneosa (type strain HTCC2155 T  = ATCC BAA‐859 T  = KCTC 12141 T ) gen. nov., sp. nov., the cultured marine representative of the Lentisphaerae phyl. nov., and the phylum be divided into two novel orders named the Lentisphaerales ord. nov. and the Victivallales ord. nov.

Three Gram‐negative, rod‐shaped bacteria that were found intracellularly in two environmental and one clinical Acanthamoeba sp. isolates were analysed. Two endocytobionts showing a parasitic behaviour were propagated successfully outside their amoebal host cells and were identified subsequently by comparative 16S rRNA sequence analysis as being most closely affiliated with Flavobacterium succinicans (99% 16S rRNA sequence similarity) or Flavobacterium johnsoniae (98% 16S rRNA sequence similarity). One endocytobiont could neither be cultivated outside its original Acanthamoeba host ( Acanthamoeba sp. TUMSJ‐321) nor transferred into other amoebae. Electron microscopy revealed that the amoebal trophozoites and cysts were almost completely filled with cells of this endosymbiont which are surrounded by a host‐derived membrane. According to 16S rRNA sequence analysis, this endosymbiont could also be assigned to the Cytophaga – Flavobacterium – Bacteroides (CFB) phylum, but was not closely affiliated to any recognized species within this phylogenetic group (less than 82% 16S rRNA sequence similarity). Identity and intracellular localization of this endosymbiont were confirmed by application of a specific fluorescently labelled 16S rRNA‐targeted probe. Based on these findings, we propose classification of this obligate Acanthamoeba endosymbiont as ‘ Candidatus Amoebophilus asiaticus’. Comparative 18S rRNA sequence analysis of the host of ‘ Candidatus Amoebophilus asiaticus’ revealed its membership with Acanthamoeba 18S rDNA sequence type T4 that comprises the majority of all Acanthamoeba isolates.

Acanthamoebae are increasingly being recognized as hosts for obligate bacterial endosymbionts, most of which are presently uncharacterized. In this study, the phylogeny of three Gram‐negative, rod‐shaped endosymbionts and their Acanthamoeba host cells was analysed by the rRNA approach. Comparative analyses of 16S rDNA sequences retrieved from amoebic cell lysates revealed that the endosymbionts of Acanthamoeba polyphaga HN‐3, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39 are related to the Paramecium caudatum endosymbionts Caedibacter caryophilus, Holospora elegans a n d Holospora obtusa . With overall 16S rRNA sequence similarities to their closest relative, C. caryophilus , of between 87% and 93%, these endosymbionts represent three distinct new species. In situ hybridization with fluorescently labelled endosymbiont‐specific 16S rRNA‐targeted probes demonstrated that the retrieved 16S rDNA sequences originated from the endosymbionts and confirmed their intracellular localization. We propose to classify provisionally the endosymbiont of Acanthamoeba polyphaga HN‐3 as ‘ Candidatus Caedibacter acanthamoebae’, the endosymbiont of Acanthamoeba sp. strain UWC9 as ‘ Candidatus Paracaedibacter acanthamoebae’ and the endosymbiont of Acanthamoeba sp. strain UWE39 as ‘ Candidatus Paracaedibacter symbiosus’. The phylogeny of the Acanthamoeba host cells was analysed by comparative sequence analyses of their 18S rRNA. Although Acanthamoeba polyphaga HN‐3 clearly groups together with most of the known Acanthamoeba isolates (18S rRNA sequence type 4), Acanthamoeba sp. UWC9 and UWE39 exhibit < 92% 18S rRNA sequence similarity to each other and to other Acanthamoeba isolates. Therefore, we propose two new sequence types (T13 and T14) within the genus Acanthamoeba containing, respectively, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39.

A morphologically conspicuous bacterium that constituted a very small fraction (< 0.01%) of the total microbial community of activated sludge was enriched and analysed phylogenetically by a combination of cultivation‐independent molecular and physical methods. The large, corkscrew‐shaped, filamentous bacteria were first detected in municipal activated sludge by light microscopy owing to their unusual rotating gliding motility. Various attempts at microbiological enrichment and pure culture isolation with traditional techniques failed, as did attempts to retrieve the morphotype of interest by micromanipulation. In situ hybridization with the group‐specific, rRNA‐targeted oligonucleotide probe CF319a indicated a phylogenetic affiliation to the Cytophaga–Flexibacter group of the Cytophaga – Flavobacterium–Bacteroides phylum. Based on strong morphological resemblance to members of the genus Saprospira , additional 16S rRNA‐targeted oligonucleotides with more narrow specificity were designed and evaluated for in situ hybridization to the morphotype of interest. Flow cytometric cell sorting based on the fluorescence conferred by probe SGR1425 and forward scatter enabled a physical enrichment of the helical coiled cells. Subsequent polymerase chain reaction (PCR) amplification of 16S rDNA fragments from whole fixed sorted cells with a primer pair based on probes CF319a and SGR1425 resulted in the retrieval of 12 almost identical partial 16S rDNA fragments with sequence similarities among each other of more than 99.2%. In situ hybridizations proved that the sequences that showed the highest similarity (88.4%) to the 16S rRNA of Saprospira grandis were indeed retrieved from the corkscrew‐shaped filaments. The bacterium is likely to be a member of a genus of which no species has been cultured hitherto. It was consequently tentatively named ‘Magnospira bakii’ and has the taxonomic rank of Candidatus Magnospira bakii, as the ultimate taxonomic placement has to await its cultivation. In this study, it was demonstrated that even bacteria occurring at very low frequencies in highly complex environmental samples can be retrieved selectively without cultivation for further molecular analysis.