Search

Fluorescence in situ hybridization (FISH) has impacted profoundly on our knowledge of the in situ ecophysiology and biodiversity of bacteria in natural communities. However, it has many technical challenges including the possibility of false positives from the binding of probes to non-target rRNA sequences. We show here that probe target sites containing single-base insertions or deletions can lead to false FISH positives, the result of hybridization with a bulge around the missing base. Experimental and in silico data suggest this situation occurs at a surprisingly high frequency. The existence of such sites is not currently considered during most FISH probe design processes. We describe software to identify potential non-target sites resulting from single-base insertions or deletions in rRNA sequences. This software also provides an estimate of the FISH probe hybridization efficiency to these sites., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The in situ physiology of the filamentous bacterium Skermania piniformis frequently seen in activated sludge foams in Australia was investigated. An oligonucleotide probe, Spin1449, targeting the 16S rRNA of S. piniformis was designed for its identification by fluorescence in situ hybridization (FISH), validated with pure cultures and applied successfully to foam samples from two geographically distant Australian plants. While filaments of this bacterium appeared to be comparatively hydrophobic, the organism had no clear preference for hydrophobic or hydrophilic substrates. In both foams examined using microautoradiography (MAR), filaments selectively took up substrates under aerobic and anoxic (NO(3) (-)) but not anaerobic or anoxic (NO(2) (-)) conditions. Skermania piniformis assimilated oleic acid, palmitic acid, glycerol and glycine. Ectoenzyme activities detected suggest that S. piniformis has an ability to assimilate a greater range of substrates than might be concluded from the MAR data obtained here. Based on the substrate uptake data presented here, an anaerobic selector may work for controlling S. piniformis in activated sludge systems.

16S rRNA targeted probes, designed using sequence data from pure cultures of the three morphotypes of the filamentous bulking bacteria Nostocoida limicola I, II and III and their successful application to the in situ identification of these bacteria in activated sludge biomass samples are described here. Two probes were required to detect all the sequenced N. limicola II isolates. Results from fluorescent in situ hybridization suggest that the morphotypes N. limicola I and II contain at least two phylogenetically unrelated bacteria. The N. limicola II filaments that did not respond to the probes designed in this study fluoresced instead with the probes previously designed for the alpha-Proteobacteria. The data also suggest that both N. limicola I and III can exist in activated sludge as single, paired or clumped cells and thus in a form not recognizable microscopically as this morphotype. Some N. limicola II filaments which responded to the probes designed here were much thinner than the filaments conventionally 'identified' as this morphotype and better fitted the descriptions often used in the literature for N. limicola I.

This review discusses a group of bacteria, the 'G-bacteria', which have a distinctive morphology of cocci in tetrads, sheets or clusters, that are seen in large numbers in many activated sludge biomass samples. Isolates of 'G-bacteria' that have been grown axenically are phylogenetically diverse. The Gram-negative members include several alpha- and beta-proteobacteria, among which is the genus Amaricoccus, while the Gram-positive 'G-bacteria' contain several members of the actinobacteria. It is probable that other, as yet uncharacterized, 'G-bacteria' exist in activated sludge. The hypothesis that these 'G-bacteria' are detrimental to the process of enhanced biological phosphate removal by competing for substrates anaerobically with the phosphate-accumulating bacteria in such systems, based as it is largely on mixed-culture studies, receives little support from studies using those available in pure culture. The evidence on which these conclusions are founded is discussed, as are the arguments used to explain why these 'G-bacteria' all appear to thrive under conditions found in certain activated sludge systems.