Preface to the Princeton Landmarks in Biology Edition vii Preface xi Symbols Used xiii 1. The Importance of Islands 3 2. Area and Number of Speicies 8 3. Further Explanations of the Area-Diversity Pattern 19 4. The Strategy of Colonization 68 5. Invasibility and the Variable Niche 94 6. Stepping Stones and Biotic Exchange 123 7. Evolutionary Changes Following Colonization 145 8. Prospect 181 Glossary 185 References 193 Index 201

The Theory of Island Biogeography

Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

Publisher Summary Recent advances in the biochemistry of microorganisms have revealed that cell-component analysis can be effectively applied to bacterial systematics, providing the basis of chemotaxonomy. Analyses of cell components are coming to be essential tools not only for bacterial classification but also for identification. The sequence of chromosomal DNA holds the essential genetic information for an organism. Phylogenetic relationships are often examined using data on nucleotide sequences of ribosomal RNA. However, it is still impossible to determine the sequences of large polynucleotides for the number of organisms sufficient for taxonomic purposes. DNA (RNA) homologies, which have been applied to various kinds of bacteria, present useful information on bacterial systematics. Homology indices are values used in comparing microorganisms. DNA homology data contribute to the clarification, not only of the relatedness of two organisms, but also of the evaluation of phenotypic characteristics.

4 Lipid and Cell-Wall Analysis in Bacterial Systematics

Most natural environments harbour a stunningly diverse collection of microbial species. In these communities, bacteria compete with their neighbours for space and resources. Laboratory experiments with pure and mixed cultures have revealed many active mechanisms by which bacteria can impair or kill other microorganisms. In addition, a growing body of theoretical and experimental population studies indicates that the interactions within and between bacterial species can have a profound impact on the outcome of competition in nature. The next challenge is to integrate the findings of these laboratory and theoretical studies and to evaluate the predictions that they generate in more natural settings.

Bacterial competition: surviving and thriving in the microbial jungle

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.

Dynamic transition of a methanogenic population in response to the concentration of volatile fatty acids in a thermophilic anaerobic digester.

Hydrogen production by thermophilic anaerobic microflora enriched from sludge compost was studied by using an artificial medium containing cellulose powder. Hydrogen gas was evolved with the formation of acetate, ethanol, and butyrate by decomposition of the cellulose powder. The hydrogen production yield was 2.0 mol/mol-hexose by either batch or chemostat cultivation. A medium that did not contain peptone demonstrated a lower hydrogen production yield of 1.0 mol/mol-hexose with less formation of butyrate. The microbial community in the microflora was investigated through isolation of the microorganisms by both plating and denaturing gradient gel electrophoresis (DGGE) of the' PCR-amplified V3 region of 16S rDNA. Sixty-eight microorganisms were isolated from the microflora and classified into nine distinct groups by genetic fingerprinting of the PCR-DGGE or by a random amplified polymorphic DNA analysis and determination of the partial sequence of 16S rDNA. Most of the isolates belonged to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G+C gram-positive bacteria. Product formation by most of the isolated strains corresponded to that produced by the microflora. Thermoanaerobacterium thermosaccharolyticium was isolated in the enrichment culture with or without added peptone. and was detected with strong intensity by PCR-DGGE. Two other thermophilic cellulolytic microorganisms, Clostridium thermocellum and Clostridium cellulosi, were also detected by PCR-DGGE, although they could not be isolated. These findings imply that hydrogen production from cellulose by microflora is performed by a consortium of several species of microorganisms.

Microbial community in anaerobic hydrogen-producing microflora enriched from sludge compost

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, "knockout communities" in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.

Stable coexistence of five bacterial strains as a cellulose-degrading community.

We bred a microbial community capable of degrading rice straw with high efficiency. The microbial community degraded more than 60% of rice straw within 4 days at 50 °C. The high stability of the community's degradation ability was demonstrated by its tolerance of being subcultured several times in medium with/without cellulosic material, being heated to 95 °C, and freezing at –80 °C. The community degraded both nonsterilized and sterilized substrate; and its degradation ability was not affected by pH changes in the medium (initial pH 5–9). PCR-denaturing gradient gel electrophoresis (DGGE) analyses based on 16S rDNA fragments showed that the community structure remained constant after multiple subcultures extending over 2 years. DNA sequence analyses of DGGE bands indicated the coexistence of both aerobic and anaerobic bacteria in the community.

Construction of a stable microbial community with high cellulose-degradation ability

In anoxic rice field soil, ferric iron reduction is one of the most important terminal electron accepting processes, yet little is known about the identity of iron-reducing microorganisms. Here, we identified acetate-metabolizing bacteria by RNA-based stable isotope probing in the presence of iron(III) oxides as electron acceptors. After reduction of endogenous iron(III) for 21 days, isotope probing with (13)C-labeled acetate (2 mM) and added ferric iron oxides (ferrihydrite or goethite) was performed in rice field soil slurries for 48 and 72 h. Ferrihydrite reduction coincided with a strong suppression of methanogenesis (77%). Extracted RNA from each treatment was density resolved by isopycnic centrifugation, and analyzed by terminal restriction fragment length polymorphism, followed by cloning and sequencing of 16S rRNA of bacterial and archaeal populations. In heavy, isotopically labeled RNAs of the ferrihydrite treatment, predominant (13)C-assimilating populations were identified as Geobacter spp. (approximately 85% of all clones). In the goethite treatment, iron(II) formation was not detectable. However, Geobacter spp. (approximately 30%), the delta-proteobacterial Anaeromyxobacter spp. (approximately 30%), and novel beta-Proteobacteria were predominant in heavy rRNA fractions indicating that (13)C-acetate had been assimilated in the presence of goethite, whereas none were detected in the control heavy RNA. For the first time, active acetate-oxidizing iron(III)-reducing bacteria, including novel hitherto unrecognized populations, were identified as a functional guild in anoxic paddy soil.

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Yasuo Igarashi

Papers

Dynamic transition of a methanogenic population in response to the concentration of volatile fatty acids in a thermophilic anaerobic digester.

Microbial community in anaerobic hydrogen-producing microflora enriched from sludge compost

Stable coexistence of five bacterial strains as a cellulose-degrading community.

Construction of a stable microbial community with high cellulose-degradation ability

Identification of iron-reducing microorganisms in anoxic rice paddy soil by 13C-acetate probing