Christine Moissl

Isolation and Characterization of Bacteria Capable of Tolerating the Extreme Conditions of Clean Room Environments

ABSTRACT In assessing the bacterial populations present in spacecraft assembly, spacecraft test, and launch preparation facilities, extremophilic bacteria (requiring severe conditions for growth) and extremotolerant bacteria (tolerant to extreme conditions) were isolated. Several cultivation approaches were employed to select for and identify bacteria that not only survive the nutrient-limiting conditions of clean room environments but can also withstand even more inhospitable environmental stresses. Due to their proximity to spacefaring objects, these bacteria pose a considerable risk for forward contamination of extraterrestrial sites. Samples collected from four geographically distinct National Aeronautics and Space Administration clean rooms were challenged with UV-C irradiation, 5% hydrogen peroxide, heat shock, pH extremes (pH 3.0 and 11.0), temperature extremes (4°C to 65°C), and hypersalinity (25% NaCl) prior to and/or during cultivation as a means of selecting for extremotolerant bacteria. Culture-independent approaches were employed to measure viable microbial (ATP-based) and total bacterial (quantitative PCR-based) burdens. Intracellular ATP concentrations suggested a viable microbial presence ranging from below detection limits to 106 cells/m2. However, only 0.1 to 55% of these viable cells were able to grow on defined culture medium. Isolated members of the Bacillaceae family were more physiologically diverse than those reported in previous studies, including thermophiles (Geobacillus), obligate anaerobes (Paenibacillus), and halotolerant, alkalophilic species (Oceanobacillus and Exiguobacterium). Non-spore-forming microbes (α- and β-proteobacteria and actinobacteria) exhibiting tolerance to the selected stresses were also encountered. The multiassay cultivation approach employed herein enhances the current understanding of the physiological diversity of bacteria housed in these clean rooms and leads us to ponder the origin and means of translocation of thermophiles, anaerobes, and halotolerant alkalophiles into these environments.

The unique structure of archaeal 'hami', highly complex cell appendages with nano-grappling hooks

Proteinaceous, hair‐like appendages known as fimbriae or pili commonly extend from the surface of prokaryotic cells and serve important functions such as cell adhesion, biofilm formation, motility and DNA transfer. Here we show that a novel group of archaea from cold, sulphidic springs has developed cell surface appendages of an unexpectedly high complexity with a well‐defined base‐to‐top organization. It represents a new class of filamentous cell appendages, for which the term ‘hamus’ is proposed. Each archaeal cell is surrounded by a halo of about 100 hami, which mediate strong adhesion of the cells to surfaces of different chemical composition. The hami are mainly composed of 120 kDa subunits and remained stable in a broad temperature and pH range (0–70°C; 0.5–11.5). Electron microscopy and cryo‐electron tomography revealed that the hamus filament possesses a helical basic structure. At periodic distances, three prickles emanate from the filament, giving it the character of industrially produced barbwire. At its distal end the hami carry a tripartite, barbed grappling hook (60 nm in diameter). The architecture of this molecular hook is reminiscent of man‐made fishhooks, grapples and anchors. It appears that nature has developed a perfect mechanical nano‐tool in the course of biological evolution, which also might prove useful in the field of nanobiotechnology.

Halorhabdus tiamatea sp. nov., a non-pigmented, extremely halophilic archaeon from a deep-sea, hypersaline anoxic basin of the Red Sea, and emended description of the genus Halorhabdus.

An extremely halophilic archaeon was isolated from a sample of the brine-sediment interface of the Shaban Deep in the northern Red Sea. Phylogenetic analysis of the 16S rRNA gene sequence revealed a close proximity to Halorhabdus utahensis (99.3%), the sole species of the genus Halorhabdus. Strain SARL4B(T) formed non-pigmented colonies and showed optimum growth at 45 degrees C, in 27% (w/v) NaCl and at pH 6.5-7.0. This organism utilized a few complex substrates, such as yeast extract and starch, for growth. Strain SARL4B(T) grew under anaerobic and microaerophilic conditions but grew extremely poorly under aerobic conditions. The ether lipids were diphytanyl derivatives. The DNA G+C content of the type strain was 61.7 mol%. On the basis of the phylogenetic data and physiological and biochemical characteristics, strain SARL4B(T) represents a novel species of the genus Halorhabdus, for which the name Halorhabdus tiamatea is proposed. The type strain is SARL4B(T) (=DSM 18392(T)=JCM 14471(T)). An emended description of the genus Halorhabdus is also proposed.

Natural Communities of Novel Archaea and Bacteria with a String-of-Pearls-Like Morphology: Molecular Analysis of the Bacterial Partners

ABSTRACT A recently discovered bacterial/archaeal association, growing in a string-of-pearls-like structure, thrives in the cold (∼10°C) sulfidic marsh water of the Sippenauer Moor near Regensburg, Bavaria, Germany. It forms characteristic, macroscopically visible globules, the pearls, containing microcolonies of novel euryarchaeota, which are surrounded by mainly filamentous bacteria (C. Rudolph, G. Wanner, and R. Huber, Appl. Environ. Microbiol. 67:2336-2344, 2001). Single pearls in series are connected by white threads. Here we report the first detailed molecular investigations of the taxonomic affiliation of the bacteria contributing to the strings of pearls. Phylogenetic analysis showed the dominance of a single phylotype (clone sipK4) within single pearls most closely related to Thiothrix unzii. The presence of Thiothrix sipK4 as a major constituent of single pearls and of the pearl-connecting white threads was verified by fluorescence in situ hybridization.

Ecology and microbial structures of archaeal/bacterial strings‐of‐pearls communities and archaeal relatives thriving in cold sulfidic springs

Recently, a unique microbial community, growing in a whitish, macroscopically visible strings-of-pearls-like structure was discovered in the cold, sulfidic marsh water of the Sippenauer Moor near Regensburg, Bavaria, Germany. The pearls interior is predominated by microcolonies of the non-methanogenic SM1 euryarchaeon; the outer part of the pearls is mainly composed of Thiothrix. To screen sulfidic ecosystems for the distribution of such unique microbial communities, comparative microbial and geochemical analyses of cold, sulfidic springs of three geographically distinct locations in Bavaria, Germany, and Dalyan, Turkey, were performed. Here, we report on the discovery and study of another type of strings-of-pearls revealing a new microbial community structure. While the SM1 euryarchaeon is again the predominant archaeal constituent, the bacterial partner is the so-called IMB1 eta-proteobacterium. Due to the predominance of the IMB1 eta-proteobacterium, the strings-of-pearls reveal a fluffy and greyish macroscopical appearance. The phylogenetic survey revealed SM1 euryarchaeal relatives, designated as SM1 group, in all sites studied, indicating a widespread distribution of these archaea in terrestrial ecosystems.

Archaeal diversity analysis of spacecraft assembly clean rooms.

One of the main tasks of NASAs planetary protection program is to prevent the forward contamination of extraterrestrial environments with Earth life, and in turn preserve other planets and the integrity of future life detection missions. Despite information regarding bacterial diversity in NASAs clean rooms, little is known about the presence of Archaea. Archaeal community analysis of spacecraft-associated surfaces is important, as they are considered by some to represent terrestrial life most capable of surviving on Mars. The first insights into the archaeal diversity of clean rooms where spacecraft assembled are attempted. Nucleic acid sequences clustering with uncultivable Archaea within the Eury- and Crenarchaeota were retrieved from 8 of 26 samples collected from several spacecraft assembly clean rooms. Due to their potential capability to survive and proliferate in Martian conditions, screening for Archaea on spacecraft surfaces and instruments that are associated with future life detection missions may be necessary.

New Insights into the Lifestyle of the Cold-Loving SM1 Euryarchaeon: Natural Growth as a Monospecies Biofilm in the Subsurface

ABSTRACT In the surface waters of sulfidic springs near Regensburg, Bavaria, Germany, the SM1 euryarchaeon, together with filamentous bacteria, forms the recently described unique string-of-pearls community. In addition to naturally occurring string-of-pearls communities, the growth of these communities was also observed on polyethylene nets provided as an artificial attachment material in the streamlets of springs. In order to learn more about the distribution and origin of the SM1 euryarchaeon and its possible occurrence in the subsurface, polyethylene nets were incubated as deeply as possible in different spring holes. After a short residence time, slime-like, milky drops, almost completely composed of SM1 euryarchaeon, were attached to the nets, indicating that this organism grows independent of a partner in deeper earth layers. A newly designed in situ biofilm trapping system allowed the quantitative harvesting of organisms exhibiting this newly discovered lifestyle of the SM1 euryarchaeon for detailed biological studies. The discovery of naturally occurring archaeal biofilms extends our knowledge of the biology and ecological significance of archaea in their environments.