Constraining the interpretation of 2-methylhopanoids through genetic and phylogenetic methods

Hopanoids are a class of sterol-like lipids produced by select bacteria. Their preservation in the rock record for billions of years as fossilized hopanes lends them geological significance. Much of the structural diversity present in this class of molecules, which likely underpins important biological functions, is lost during fossilization. Yet, one type of modification that persists during preservation is methylation at C-2. The resulting 2-methylhopanoids are prominent molecular fossils and have an intriguing pattern over time, exhibiting increases in abundance associated with Ocean Anoxic Events during the Phanerozoic. This thesis uses diverse methods to address what the presence of 2-methylhopanes tells us about the microbial life and environmental conditions of their ancient depositional settings. Through an environmental survey of hpnP, the gene encoding the C-2 hopanoid methylase, we found that many different taxa are capable of producing 2-methylhopanoids in more diverse modern environments than expected. This study also revealed that hpnP is significantly overrepresented in organisms that are plant symbionts, in environments associated with plants, and with metabolisms that support plant-microbe interactions; collectively, these correlations provide a clue about the biological importance of 2-methylhopanoids. Phylogenetic reconstruction of the evolutionary history of hpnP revealed that 2-methylhopanoid production arose in the Alphaproteobacteria, indicating that the origin of these molecules is younger than originally thought. Additionally, we took genetic approach to understand the role of 2-methylhopanoids in Cyanobacteria using the filamentous symbiotic Nostoc punctiforme. We found that hopanoids likely aid in rigidifying the cell membrane but do not appear to provide resistance to osmotic or outer membrane stressors, as has been shown in other organisms. The work presented in this thesis supports previous findings that 2-methylhopanoids are not biomarkers for oxygenic photosynthesis and provides new insights by defining their distribution in modern environments, identifying their evolutionary origin, and investigating their role in Cyanobacteria. These efforts in modern settings aid the formation of a robust interpretation of 2-methylhopanes in the rock record.

The microbiological quality of water: the nature of the problem

Improvements in methods for the detection and enumeration of microbes in water, particularly the application of techniques of molecular biology, have highlighted shortcomings in the ”standard methods” for assessing water quality. Higher expectations from the consumer and increased publicity associated with pollution incidents can lead to an uncoupling of the cycle which links methodological development with standard-setting and legislation. The new methodology has also highlighted problems within the water cycle, related to the introduction, growth and metabolism of microbes. A greater understanding of the true diversity of the microbial community and the ability to transmit genetic information within aquatic systems ensures that the subject of this symposium and volume provides an ideal forum to discuss the problems encountered by both researcher and practitioner.

The impact of molecular biology on assessment of water quality: advantages and limitations of current techniques

The advent of molecular biology has had a dramatic impact on all aspects of biology, not least applied microbial ecology. Microbiological testing of water has traditionally depended largely on culture techniques. Growing understanding that only a small proportion of microbial species are culturable, and that many microorganisms may attain a viable but non-culturable state, has promoted the development of novel approaches to monitoring pathogens in the environment. This has been paralleled by an increased awareness of the surprising genetic diversity of natural microbial populations. By targeting gene sequences that are specific for particular microorganisms, for example genes that encode diagnostic enzymes, or species-specific domains of conserved genes such as 16S ribosomal RNA coding sequences (rrn genes), the problems of culture can be avoided. Technical developments, notably in the area of in vitro amplification of DNA using the polymerase chain reaction (PCR), now permit routine detection and identification of specific microorganisms, even when present in very low numbers. Although the techniques of molecular biology have provided some very powerful tools for environmental microbiology, it should not be forgotten that these have their own drawbacks and biases in sampling. For example, molecular techniques are dependent on efficient lysis and recovery of nucleic acids from both vegetative forms and spores of microbial species that may differ radically when growing in the laboratory compared with the natural environment. Furthermore, PCR amplification can introduce its own bias depending on the nature of the oligonucleotide primers utilised. However, despite these potential caveats, it seems likely that a molecular biological approach, particularly with its potential for automation, will provide the mainstay of diagnostic technology for the foreseeable future.

Human LDL Structural Diversity Studied by IR Spectroscopy

Lipoproteins are responsible for cholesterol traffic in humans. Low density lipoprotein (LDL) delivers cholesterol from liver to peripheral tissues. A misleading delivery can lead to the formation of atherosclerotic plaques. LDL has a single protein, apoB-100, that binds to a specific receptor. It is known that the failure associated with a deficient protein-receptor binding leads to plaque formation. ApoB-100 is a large single lipid-associated polypeptide difficulting the study of its structure. IR spectroscopy is a technique suitable to follow the different conformational changes produced in apoB-100 because it is not affected by the size of the protein or the turbidity of the sample. We have analyzed LDL spectra of different individuals and shown that, even if there are not big structural changes, a different pattern in the intensity of the band located around 1617 cm 21 related with strands embedded in the lipid monolayer, can be associated with a different conformational rearrangement that could affect to a protein interacting region with the receptor.

Effects of trawling for ocean shrimp (Pandalus jordani) on macroinvertebrate abundance and diversity at four sites near Nehalem Bank, Oregon

Surveys with a remotely operated vehicle (ROV) at four mudhabitat sites with different histories of ocean shrimp (Pandalus jordani) trawling showed measurable effects of trawling on macroinvertebrate abundance and diversity. Densities of the sea whip (Halipteris spp., P<0.01), the flat mud star (Luidia foliolata, P< 0.001), unidentified Asteroidea (P<0.05), and squat lobsters (unidentified Galathoidea, P<0.001) were lower at heavily trawled (HT) sites, as was invertebrate diversity based on the Shannon-Wiener index. Sea cucumbers (unidentified Holothuroidea) and unidentified corals (Hydrocoralia) were observed at lightly trawled (LT) sites but not at HT sites. Hagfish (Eptatretus spp.) burrows were the dominant structural feature of the sediment surface at all sites and were more abundant at the HT sites (P<0.05), a result potentially related to effects from fishery discards. Substantial heterogeneity was found between the northern and southern site pairs, indicating high site-to-site variability in macroinvertebrate densities in these deep (146–156 m) mud habitats. Two of the study sites were closed to trawling in June 2006. The data from this study can be used in combination with future surveys to measure recovery rates of deep, mud, seaf loor habitats from the effects of trawling, thus providing a critical piece of information for ecosystem-based management.

Emerging patterns of species richness, diversity, population density, and distribution in the skates (Rajidae) of Alaska

Six years of bottom-trawl survey data, including over 6000 trawls covering over 200 km2 of bottom area throughout Alaska’s subarctic marine waters, were analyzed for patterns in species richness, diversity, density, and distribution of skates. The Bering Sea continental shelf and slope, Aleutian Islands, and Gulf of Alaska regions were stratified by geographic subregion and depth. Species richness and relative density of skates increased with depth to the shelf break in all regions. The Bering Sea shelf was dominated by the Alaska skate (Bathyraja parmifera), but species richness and diversity were low. On the Bering Sea slope, richness and diversity were higher in the shallow stratum, and relative density appeared higher in subregions dominated by canyons. In the Aleutian Islands and Gulf of Alaska, species richness and relative density were generally highest in the deepest depth strata. The data and distribution maps presented here are based on species-level data collected throughout the marine waters of Alaska, and this article represents the most comprehensive summary of the skate fauna of the region published to date.

Diversity of estuarine movements of striped bass (Morone saxatilis): a synoptic examination of an estuarine system in southern New Jersey

We determined the dis-tribution of multiple (n=68; 508−978 mm total length [TL]) striped bass (Morone saxatilis) along the estua-rine salinity gradient in the Mullica River−Great Bay in southern New Jersey over two years to determine the diversity of habitat use and the movements of striped bass. Ultrasoni-cally tagged fish were detected in this estuarine area by means of wireless hydrophones deployed at four gates inside the entrance of the study area and farther up to tidal freshwater (38 km). Numerous individuals frequently departed and returned to the estuary, primarily in the spring and late fall over periods of 15−731 days at liberty. The period of residency and degree of movement of individuals to and from the estuary varied extensively among seasons and years. The diversity of movements in and out of, as well as within, the estuary differed from the less-complex patterns reported in earlier studies, perhaps because of the comprehensive and synoptic nature of this study.