Brendan P. Burns

Advances in on-line drinking water quality monitoring and early warning systems

Significant advances have been made in recent years in technologies to monitor drinking water quality for source water protection, treatment operations, and distribution system management, in the event of accidental (or deliberate) contamination. Reports prepared through the Global Water Research Coalition (GWRC) and United States Environment Protection Agency (USEPA) agree that while many emerging technologies show promise, they are still some years from being deployed on a large scale. Further underpinning their viability is a need to interpret data in real time and implement a management strategy in response. This review presents the findings of an international study into the state of the art in this field. These results are based on visits to leading water utilities, research organisations and technology providers throughout Europe, the United States and Singapore involved in the development and deployment of on-line monitoring technology for the detection of contaminants in water.

Phylogeography of the invasive cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a planktonic freshwater cyanobacterium that has become increasingly prevalent in tropical and temperate water bodies world‐wide. This species is of concern from a water‐quality perspective because of its known ability to produce toxins that can affect the health of humans and other animals. This study investigates genetic vari‐ation between strains of C. raciborskii isolated from freshwater rivers and reservoirs in Australia, Brazil, Germany, Hungary, Portugal and the USA. Strains were first characterized by analysis of their 16S rRNA gene nucleotide sequences and were found to have a sequence divergence of 99.1%. A phylogenetic tree, constructed using the 16S rRNA gene sequences showed that strains grouped into Australian, European and North/South American phylotypes. To investigate further the observed separation of strains into geographically distinct groups, we applied a cyanobacterium‐specific short tandem repeat sequence technique, HIP1. An electrophoretic comparison of the HIP1 polymerase chain reaction products showed clear distinctions between the C. raciborskii strains. A phylogenetic tree, based on the repeat element banding patterns, also revealed three distinct groups of C. raciborskii strains. The first group consisted of strains from the USA and Brazil; the second comprised European strains from Germany, Hungary and Portugal; and the third were strains from Australia. In general, between‐country variation was greater than within‐country variation, indicating that this fingerprinting technique can successfully distinguish C. raciborskii strains taken from different global locations. The relationship between toxicity and the observed HIP1 polymerase chain reaction fingerprint profiles was less clear, although it is interesting to note that of the strains analysed in this study, only Australian strains are known to produce cylindrospermopsin and only Brazilian strains have been reported to produce paralytic shellfish poisoning toxins.

Detection and sequencing of the microcystin LR-degrading gene, mlrA, from new bacteria isolated from Japanese lakes

mlrA is the only microcystin-degrading gene detected in Sphingomonas sp. MJ-PV. The gene has an extremely rare nucleotide sequence and homologous genes have not yet been discovered in the DNA database. We discovered the existence of a gene homologous to mlrA in new microcystin-degrading bacteria, MD-1 and Y2. These strains possessed mlrA homologues, and the identities of the genes of MD-1 and Y2 with the corresponding MJ-PV exceeded 98% and 84%, respectively. On the other hand, the mlrA gene was not detected in laboratory strains of the closely related Sphingomonas spp. strains employing hemi-nested polymerase chain reaction detection using two primer sets. Although the microcystin-degrading bacteria were closely related strains, they did not cluster together as the same species. We can conclude that the mlrA gene is conserved in three different bacterial species, and it is unique to microcystin degraders but not to the genus Sphingomonas.

Bacterial, archaeal and eukaryotic diversity of smooth and pustular microbial mat communities in the hypersaline lagoon of Shark Bay.

The bacterial, archaeal and eukaryotic populations of nonlithifying mats with pustular and smooth morphology from Hamelin Pool, Shark Bay were characterised using small subunit rRNA gene analysis and microbial isolation. A highly diverse bacterial population was detected for each mat, with 16S rDNA clones related to Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Gemmatimonas, Planctomycetes, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Verrucomicrobia and candidate division TM6 present in each mat. Spirochaetes were detected in the smooth mat only, whereas candidate division OP11 was only detected in the pustular mat. Targeting populations with specific primers revealed additional cyanobacterial diversity. The archaeal population of the pustular mat was comprised purely of Halobacteriales, whereas the smooth mat contained 16S rDNA clones from the Halobacteriales, two groups of Euryarchaea with no close characterised matches, and the Thaumarchaea. Nematodes and fungi were present in each mat type, with diatom 18S rDNA clones only obtained from the smooth mat, and tardigrade and microalgae clones only retrieved from the pustular mat. Cultured isolates belonged to the Firmicutes, Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, and Halobacteriales. The mat populations were significantly more diverse than those previously reported for Hamelin Pool stromatolites, suggesting specific microbial populations may be associated with the nonlithifying and lithifying microbial communities of Hamelin Pool.

Determining the specific microbial populations and their spatial distribution within the stromatolite ecosystem of Shark Bay

The stromatolites at Shark Bay, Western Australia, are analogues of some of the oldest evidence of life on Earth. The aim of this study was to identify and spatially characterize the specific microbial communities associated with Shark Bay intertidal columnar stromatolites. Conventional culturing methods and construction of 16S rDNA clone libraries from community genomic DNA with both universal and specific PCR primers were employed. The estimated coverage, richness and diversity of stromatolite microbial populations were compared with earlier studies on these ecosystems. The estimated coverage for all clone libraries indicated that population coverage was comprehensive. Phylogenetic analyses of stromatolite and surrounding seawater sequences were performed in ARB with the Greengenes database of full-length non-chimaeric 16S rRNA genes. The communities identified exhibited extensive diversity. The most abundant sequences from the stromatolites were α- and γ-proteobacteria (58%), whereas the cyanobacterial community was characterized by sequences related to the genera Euhalothece, Gloeocapsa, Gloeothece, Chroococcidiopsis, Dermocarpella, Acaryochloris, Geitlerinema and Schizothrix. All clones from the archaeal-specific clone libraries were related to the halophilic archaea; however, no archaeal sequence was identified from the surrounding seawater. Fluorescence in situ hybridization also revealed stromatolite surfaces to be dominated by unicellular cyanobacteria, in contrast to the sub-surface archaea and sulphate-reducing bacteria. This study is the first to compare the microbial composition of morphologically similar stromatolites over time and examine the spatial distribution of specific microorganismic groups in these intertidal structures and the surrounding seawater at Shark Bay. The results provide a platform for identifying the key microbial physiology groups and their potential roles in modern stromatolite morphogenesis and ecology.

Glucose utilization and lactate production by Helicobacter pylori

The transport and incorporation of D-glucose into the human pathogen Helicobacter pylori was investigated employing radioactive tracer analysis and 1H and 13C nuclear magnetic resonance spectroscopy. The bacterium was found to utilize D-glucose contrary to the accepted view that it cannot catabolize carbohydrates. Under the experimental conditions employed, the rate of transport of [14C]glucose was 3.24 mmol min-1 (g protein)-1, and the rate of incorporation into the cellular mass was 1.06 mumol h-1 (g protein)-1. The utilization of [13C]glucose showed biphasic characteristics with a slower initial period followed by a phase with a rate of utilization at least an order of magnitude faster. The apparent rates of decline of glucose levels during both phases varied between strains and depended on the growth conditions of the bacteria prior to harvesting. The main product of glucose catabolism was identified as lactate. These findings provide new perspectives into the physiology of H. pylori and have implications for the active search to develop appropriate therapies for the micro-organism.

Microgravity induces pelvic bone loss through osteoclastic activity, osteocytic osteolysis, and osteoblastic cell cycle inhibition by CDKN1a/p21.

Bone is a dynamically remodeled tissue that requires gravity-mediated mechanical stimulation for maintenance of mineral content and structure. Homeostasis in bone occurs through a balance in the activities and signaling of osteoclasts, osteoblasts, and osteocytes, as well as proliferation and differentiation of their stem cell progenitors. Microgravity and unloading are known to cause osteoclast-mediated bone resorption; however, we hypothesize that osteocytic osteolysis, and cell cycle arrest during osteogenesis may also contribute to bone loss in space. To test this possibility, we exposed 16-week-old female C57BL/6J mice (n = 8) to microgravity for 15-days on the STS-131 space shuttle mission. Analysis of the pelvis by µCT shows decreases in bone volume fraction (BV/TV) of 6.29%, and bone thickness of 11.91%. TRAP-positive osteoclast-covered trabecular bone surfaces also increased in microgravity by 170% (p = 0.004), indicating osteoclastic bone degeneration. High-resolution X-ray nanoCT studies revealed signs of lacunar osteolysis, including increases in cross-sectional area (+17%, p = 0.022), perimeter (+14%, p = 0.008), and canalicular diameter (+6%, p = 0.037). Expression of matrix metalloproteinases (MMP) 1, 3, and 10 in bone, as measured by RT-qPCR, was also up-regulated in microgravity (+12.94, +2.98 and +16.85 fold respectively, p<0.01), with MMP10 localized to osteocytes, and consistent with induction of osteocytic osteolysis. Furthermore, expression of CDKN1a/p21 in bone increased 3.31 fold (p<0.01), and was localized to osteoblasts, possibly inhibiting the cell cycle during tissue regeneration as well as conferring apoptosis resistance to these cells. Finally the apoptosis inducer Trp53 was down-regulated by −1.54 fold (p<0.01), possibly associated with the quiescent survival-promoting function of CDKN1a/p21. In conclusion, our findings identify the pelvic and femoral region of the mouse skeleton as an active site of rapid bone loss in microgravity, and indicate that this loss is not limited to osteoclastic degradation. Therefore, this study offers new evidence for microgravity-induced osteocytic osteolysis, and CDKN1a/p21-mediated osteogenic cell cycle arrest.

Molecular Identification of Cyanobacteria Associated with Stromatolites from Distinct Geographical Locations

Modern stromatolites represent a significant resource for studying microbial ecology and evolution. A preliminary investigation was undertaken employing specific genetic probes to characterize the cyanobacteria responsible for stromatolite construction in a range of environments, including microbial mats found in Australia not previously examined with molecular methods. Isolates of cyanobacteria were collected from stromatolites in thermal springs, hypersaline lakes, and oceanic fringes on two continents. A polymerase chain reaction specific for DNA of cyanobacterial 16S rRNA was developed, the resulting products of the DNA amplification reaction were sequenced, and the data were used to infer relatedness between the isolates studied and other members of the cyanobacterial radiation. Complete sequence was generated for the region from position 27 to 408 for 13 strains of cyanobacteria associated with stromatolites. All stromatolite-derived sequences were most closely related to cyanobacteria, as indicated by local sequence alignment. It was possible to correlate genetic identity with morphological nomenclatures and to expand the phylogeny of benthic cyanobacteria. These inferences were also expanded to temporal variation in the dominant resident cyanobacterial species based on sampling of surface and core sinter laminations. Under the methods employed, only one cyanobacterial strain was detected in each sample, suggesting the possible dominance of a specific clonal population of cyanobacteria at any one time in the biota of the samples tested. The data indicate that internal core samples of a stromatolite at least 10 years old can be successfully analyzed by DNA-based methods to identify preserved cyanobacteria.

Acetyl-CoA carboxylase activity in Helicobacter pylori and the requirement of increased CO2 for growth.

A biotinylated acetyl-CoA carboxylase from the microaerophilic bacterium Helicobacter pylori was partially purified and characterized. The approximate molecular mass of the native enzyme was estimated at 235 kDa by native PAGE. A single band corresponding to approximately 24 kDa was detected by SDS-PAGE, suggesting that the native enzyme is a multi-protein complex. The protein was isolated from the soluble fraction of the cell. Catalytic activity was acetyl-CoA-dependent and inhibited by avidin but unaffected by avidin pretreated with excess biotin. The end-product of the reaction was identified as malonyl-CoA and the reaction was shown to be reversible by NMR spectroscopy. The activity of the enzyme was 0.29 mumol min-1 (mg protein)-1. The Vmax for bicarbonate was calculated at 0.73 mumol min-1 (mg protein)-1, and the affinity of the enzyme for this substrate was relatively low, with an apparent Km of 16.6 mM. These data provide the first evidence of a possible physiological role for the requirement of high levels of CO2 for growth in vitro of this bacterium.

Bioastronautics: The Influence of Microgravity on Astronaut Health

For thousands of years different cultures around the world have assigned their own meaning to the Universe. Through research and technology, we have begun to understand the nature and mysteries of the Cosmos. Last year marked the 40(th) anniversary of our first steps on the Moon, and within two decades it is hoped that humankind will have established a settlement on Mars. Space is a harsh environment, and technological advancements in material science, robotics, power generation, and medical equipment will be required to ensure that astronauts survive interplanetary journeys and settlements. The innovative field of bioastronautics aims to address some of the medical issues astronauts encounter during space travel. Astronauts are faced with several health risks during both short- and long-duration spaceflight due to the hostile environment presented in space. Some of these health problems include bone loss, muscle atrophy, cardiac dysrhythmias, and altered orientation. This review discusses the effects of spaceflight on living organisms, in particular, the specific effects of microgravity on the human body and possible countermeasures to these effects.