Comparison of DNA Extraction Efficiency and Reproducibility of Different Aeration Diffuser Biofilms Using Bead-Beating Protocol

An existing bead-beating DNA extraction protocol was employed to compare the DNA extraction recovery and fragment quality of 6 different aeration diffuser biofilms. <i>Escherichia coli</i>, <i>Gordonia amarae</i>, and mixed liquor were used as controls. The fraction of total DNA_biofilm decreased monotonically with increasing number of beat beatings (BB) when the amount of DNA present was sufficient (&#x3e;4 μg_DNA/cm²), excluding the ceramic disk. While controls required only 2 BBs, 3 out of 5 BBs achieved ≥70% of total DNA (70.3 ± 1.7%) for 5 out of 6 biofilms. Quantitative polymerase chain reaction (PCR) analyses of 353 and 1,505 basepair (bp) amplicons from pure culture extracts showed target copy numbers were not degraded for the first 2 BBs, but the third BB decreased amplicon concentrations by 0.65 and 1.12 log for <i>E. coli</i>, and 0.39 and 0.40 log for <i>G. amarae</i>, respectively. The 353 bp fragment amplification from biofilm samples showed minimal degradation for the first 3 BBs. PCR and gel electrophoresis confirmed integrity of amplified 1,505 bp DNA fragments over the 5 BBs, except in the EDPM (75 mm diameter, tube) diffuser biofilm (4.98 ± 0.62 μg_DNA/cm²). Taken together, this study showed type of diffuser membrane biofilms had no effects on extraction efficiency, but low DNA concentrations reduced extraction performance.

Interaction of Operational and Physicochemical Factors Leading to Gordonia amarae-Like Foaming in an Incompletely Nitrifying Activated Sludge Plant

ABSTRACTThe overgrowth ofGordonia amarae-like bacteria in the mixed liquor of an incompletely nitrifying water reclamation plant was inversely correlated with temperature (r= −0.78;P< 0.005) and positively correlated with the solids retention time (SRT) obtained a week prior to sampling (r= 0.67;P< 0.005). Drops followed by spikes in the food-to-mass ratio (0.18 to 0.52) and biochemical oxygen demand concentrations in primary effluent (94 to 298 mg liter−1) occurred at the initiation ofG. amarae-like bacterial growth. The total bacterial concentration did not increase as concentrations ofG. amarae-like cells increased, but total bacterial cell concentrations fluctuated in a manner similar to that ofG. amarae-like bacteria in the pseudo-steady state. The ammonium ion removal rate (percent) was inversely related toG. amarae-like cell concentrations during accelerated growth and washout phases. The dissolved oxygen concentration decreased as theG. amarae-like cell concentration decreased. The concentrations ofG. amarae-like cells peaked (2.47 × 109cells liter−1) approximately 1.5 months prior to foaming. Foaming occurred during the late pseudo-steady-state phase, when temperature declines reversed. These findings suggested that temperature changes triggered operational and physicochemical changes favorable to the growth ofG. amarae-like bacteria. Fine-scale quantitative PCR (qPCR) monitoring at weekly intervals allowed a better understanding of the factors affecting this organism and indicated that frequent sampling was required to obtain statistical significance with factors changing as the concentrations of this organism increased. Furthermore, the early identification ofG. amarae-like cells when they are confined to mixed liquor (107cells liter−1) allows management strategies to prevent foaming.

Gordonia humi sp. nov., isolated from soil

A Gram-stain-positive, non-endospore-forming actinobacterium (CC-12301T) was isolated from soil attached to a spawn used in the laboratory to grow the edible mushroom Agaricus brasiliensis. Based on 16S rRNA gene sequence similarities, strain CC-12301T was shown to belong to the genus Gordonia and was most closely related to the type strains of Gordonia hydrophobica (97.6 % similarity), Gordonia terrae (97.5 %), Gordonia amarae (97.5 %) and Gordonia malaquae (97.4 %). The quinone system was determined to consist predominantly of menaquinone MK-9(H2), minor amounts of MK-8(H2) and MK-7(H2). The polar lipid profile consisted of the major compounds diphosphatidylglycerol and phosphatidylethanolamine, moderate amounts of two phosphatidylinositol mannosides and phosphatidylinositol and minor amounts of phosphatidylglycerol, three unidentified glycolipids, two phosphoglycolipids and a phospholipid. Mycolic acids were present. These chemotaxonomic traits and the major fatty acids, which were C16 : 1 cis9, C16 : 0, C18 : 1 and tuberculostearic acid (10-methyl C18 : 0), supported the affiliation of strain CC-12301T to the genus Gordonia. The results of physiological and biochemical tests allowed clear phenotypic differentiation of strain CC-12301T from the most closely related Gordonia species. Strain CC-12301T therefore represents a novel species, for which the name Gordonia humi sp. nov. is proposed, with the type strain CC-12301T (=DSM 45298T =CCM 7727T).

Widespread Abundance of Functional Bacterial Amyloid in Mycolata and Other Gram-Positive Bacteria

ABSTRACT Until recently, extracellular functional bacterial amyloid (FuBA) has been detected and characterized in only a few bacterial species, including Escherichia coli, Salmonella, and the gram-positive organism Streptomyces coelicolor. Here we probed gram-positive bacteria with conformationally specific antibodies and revealed the existence of FuBA in 12 of 14 examined mycolata species, as well as six other distantly related species examined belonging to the phyla Actinobacteria and Firmicutes. Most of the bacteria produced extracellular fimbriae, sometimes copious amounts of them, and in two cases large extracellular fibrils were also produced. In three cases, FuBA was revealed only after extensive removal of extracellular material by saponification, indicating that there is integrated attachment within the cellular envelope. Spores of species in the genera Streptomyces, Bacillus, and Nocardia were all coated with amyloids. FuBA was purified from Gordonia amarae (from the cell envelope) and Geodermatophilus obscurus, and they had the morphology, tinctorial properties, and β-rich structure typical of amyloid. The presence of approximately 9-nm-wide amyloids in the cell envelope of G. amarae was visualized by transmission electron microscopy analysis. We conclude that amyloid is widespread among gram-positive bacteria and may in many species constitute a hitherto overlooked integral part of the spore and the cellular envelope.

Gordonia araii sp. nov. and Gordonia effusa sp. nov., isolated from patients in Japan

Two bacterial strains, IFM 10211T and IFM 10200T, were isolated from the sputum of two Japanese patients, and were subjected to a polyphasic taxonomic study. The two strains were found to have morphological, physiological and chemotaxonomic properties that were consistent with their assignment to the genus Gordonia, except for a few chemotaxonomic characteristics. Almost complete 16S rRNA gene sequences of the two strains were determined; the data showed that they are related distantly to Gordonia amarae, Gordonia hirsuta, Gordonia hydrophobica and Gordonia sihwensis, showing 16S rRNA gene sequence similarities to the type strains of these species of 96.2–97.9 %. DNA–DNA relatedness data coupled with the combination of genotypic and phenotypic data indicated that the two strains are representatives of two novel, separate species. The names proposed to accommodate these two strains are Gordonia araii sp. nov. (type strain IFM 10211T=DSM 44811T=NBRC 100433T=JCM 12131T) and Gordonia effusa sp. nov. (type strain IFM 10200T=DSM 44810T=NBRC 100432T=JCM 12130T).

Substrate uptake by Gordonia amarae in activated sludge foams by FISH-MAR

Gordonia amarae is a right-angled branching filament belonging to the mycolic acid-containing Actinobacteria which is commonly found in many foaming activated sludge wastewater treatment plants. Although studies on different substrates as sole carbon sources by pure cultures of G. amarae have been carried out, none have examined substrate uptake by this organism in situ. Uptake of several hydrophilic and hydrophobic substrates by G. amarae was evaluated in situ using a combination of fluorescence in situ hybridization and microautoradiography. G. amarae could assimilate a range of both hydrophilic and hydrophobic substrates. From the data, G. amarae appears to be physiologically active under aerobic, anaerobic and anoxic condition (NO2 and NO3) for some substrates. This might explain why attempts to control foaming caused by G. amarae using anoxic and anaerobic selectors have been unsuccessful. This study emphasizes that bacteria can behave differently in situ to pure cultures and that it is important to evaluate the in situ physiology of these bacteria if we are to better understand their role in the wastewater treatment process.