We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

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Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

H umans have long been fascinated by the dynamism of free-flowing waters. Yet we have expended great effort to tame rivers for transportation, water supply, flood control, agriculture, and power generation. It is now recognized that harnessing of streams and rivers comes at great cost: Many rivers no longer support socially valued native species or sustain healthy ecosystems that provide important goods and services (Naiman et al. 1995, NRC 1992).

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The Natural Flow Regime

The influence of diet on the distribution of nitrogen isotopes in animals was investigated by analyzing animals grown in the laboratory on diets of constant nitrogen isotopic composition. 
The isotopic composition of the nitrogen in an animal reflects the nitrogen isotopic composition of its diet. The δ^(15)N values of the whole bodies of animals are usually more positive than those of their diets. Different individuals of a species raised on the same diet can have significantly different δ^(15)N values. The variability of the relationship between the δ^(15)N values of animals and their diets is greater for different species raised on the same diet than for the same species raised on different diets. Different tissues of mice are also enriched in ^(15)N relative to the diet, with the difference between the δ^(15)N values of a tissue and the diet depending on both the kind of tissue and the diet involved. The δ^(15)N values of collagen and chitin, biochemical components that are often preserved in fossil animal remains, are also related to the δ^(15)N value of the diet. 
The dependence of the δ^(15)N values of whole animals and their tissues and biochemical components on the δ^(15)N value of diet indicates that the isotopic composition of animal nitrogen can be used to obtain information about an animal's diet if its potential food sources had different δ^(15)N values. The nitrogen isotopic method of dietary analysis probably can be used to estimate the relative use of legumes vs non-legumes or of aquatic vs terrestrial organisms as food sources for extant and fossil animals. However, the method probably will not be applicable in those modern ecosystems in which the use of chemical fertilizers has influenced the distribution of nitrogen isotopes in food sources. 
The isotopic method of dietary analysis was used to reconstruct changes in the diet of the human population that occupied the Tehuacan Valley of Mexico over a 7000 yr span. Variations in the δ^(15)C and δ^(15)N values of bone collagen suggest that C_4 and/or CAM plants (presumably mostly corn) and legumes (presumably mostly beans) were introduced into the diet much earlier than suggested by conventional archaeological analysis.

Influence of Diet On the Distribtion of Nitrogen Isotopes in Animals

The flow regime is regarded by many aquatic ecologists to be the key driver of river and floodplain wet- land ecosystems. We have focused this literature review around four key principles to highlight the important mech- anisms that link hydrology and aquatic biodiversity and to illustrate the consequent impacts of altered flow regimes: Firstly, flow is a major determinant of physical habitat in streams, which in turn is a major determinant of biotic com- position; Secondly, aquatic species have evolved life history strategies primarily in direct response to the natural flow regimes; Thirdly, maintenance of natural patterns of longitu- dinal and lateral connectivity is essential to the viability of populations of many riverine species; Finally, the invasion and success of exotic and introduced species in rivers is facilitated by the alteration of flow regimes. The impacts of flow change are manifest across broad taxonomic groups including riverine plants, invertebrates, and fish. Despite growing recognition of these relationships, ecologists still struggle to predict and quantify biotic responses to altered flow regimes. One obvious difficulty is the ability to distin- guish the direct effects of modified flow regimes from im- pacts associated with land-use change that often accom- panies water resource development. Currently, evidence about how rivers function in relation to flow regime and the flows that aquatic organisms need exists largely as a series of untested hypotheses. To overcome these problems, aquatic science needs to move quickly into a manipulative or experimental phase, preferably with the aims of restora- tion and measuring ecosystem response.

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Basic principles and ecological consequences of altered flow regimes for aquatic biodiversity.

Hydrologic regimes play a major role in determining the biotic composition, structure, and function of aquatic, wetland, and riparian ecosystems. But human land and water uses are substantially altering hydrologic regimes around the world. Improved quantitative evaluations of human-induced hydrologic changes are needed to advance research on the biotic implications of hydrologic alteration and to support ecosystem management and restoration plans. We propose a method for assessing the degree of hydrologic alteration attributable to human influence within an ecosystem. This method, referred to as the “Indicators of Hydrologic Alteration,” is based upon an analysis of hydrologic data available either from existing measurement points within an ecosystem (such as at stream gauges or wells) or model-generated data. We use 32 parameters, organized into five groups, to statistically characterize hydrologic variation within each year. These 32 parameters provide information on ecologically significant features of surface and ground water regimes influencing aquatic, wetland, and riparian ecosystems. We then assess the hydrologic perturbations associated with activities such as dam operations, flow diversion, groundwater pumping, or intensive land-use conversion by comparing measures of central tendency and dispersion for each parameter between user-defined “pre-impact” and “post-impact” time frames, generating 64 Indicators of Hydrologic Alteration. This method is intended for use with other ecosystem metrics in inventories of ecosystem integrity, in planning ecosystem management activities, and in setting and measuring progress toward conservation or restoration goals.

A Method for Assessing Hydrologic Alteration within Ecosystems

Ecological processes in large rivers are controlled by their flow variability. However, it is difficult to find measures of hydrological variability that characterize groups of rivers and can also be used to generate hypotheses about their ecology. Multivariate analyses of the hydrographs of 52 rivers worldwide revealed distinctive patterns of flow variability that were often correlated with climate. For example, there were groups of rivers that corresponded broadly with ‘tropical’ and ‘dryland’ climates. However, some rivers from continental climates occupy both extremes of this range, illustrating the limitations of simple classification. Individual rivers and groups of rivers may also have different hydrographic ‘signatures’, and attempts to combine measures of hydrological variability into indices mask biologically significant information. This paper identifies 11 relatively independent measures of hydrological variability that help categorize river types and are each associated with aspects of fish biology. Ways are suggested by which the Flood Pulse Concept can be expanded to encompass hydrological variability and accommodate differences among groups of rivers from different climatic regions. Such recognition of the complex role of hydrological variability enhances the value of the concept for river conservation, management and restoration.

Flow variability and the ecology of large rivers

The ecosystem concept should be reappraised as a basic model for rivers, with regard for flow as an organizing variable. This would facilitate comparisons between the large rivers of humid climates, where flow regimes are comparatively regular, and those of arid and semi-arid areas, where river regimes are highly variable. Ecosystem processes might be modelled by combining the river continuum and flood pulse concepts, with refinements to accommodate a complex flood pulse (e.g. variations in stage amplitude, timing, duration, rates of rise and fall). Patch boundaries (ecotones) such as the riverine littoral zone warrant close study because they strongly influence the structure and dynamics of the ecosystem. The general model needs a quantitative basis, perhaps focused on the balance of processes involved in the physical transport and biological transformation of carbon. The ultimate test of such a model will be in its capacity to predict the effects of flow regulation. Further development, however, is limited by data. In both research and management monitoring programmes need to be established to provide information and to develop a sustained, comprehensive approach to dryland rivers as ecosystems.

A perspective on dryland river ecosystems

Selected water regime indices are used to describe the tolerances to flooding and exposure of littoral and floodplain plants of the River Murray, South Australia. The cover and abundance of 26 perennial species were surveyed at 12 sites along a reach where water levels were influenced by weir operations. Six indices were measured: days when water depths were ≥0, 0–20, 20–60 and ≥200 cm; days when plants were exposed to ≥100 cm of water; and days of longest exposure to water. Ordinations of plant abundances were correlated with the frequency of flooding to 0–20 and 20–60 cm, and exposure to ≥100 cm. Five species clusters were apparent, these being common floodplain species (e.g. Muehlenbeckia florulenta), uncommon floodplain species (e.g. Eleocharis acuta), species from the infrequently flooded littoral (e.g. Bolboschoenus caldwellii), species from the permanently flooded littoral (e.g. Vallisneria americana) and widespread, common species tolerant to flooding and exposure (11 species, including Phragmites australis, Cyperus gymnocaulos and Bolboschoenus medianus). Half of the 26 species occurred in at least four of seven regimes suggested by cluster analysis of water regime indices, thus indicating a broad tolerance to flooding and exposure. Preferred water regimes are summarised using minimum and maximum values and quartiles for the six indices, and similarities between preferences are illustrated by a model based on minimum spanning tree techniques. Copyright © 1999 John Wiley & Sons, Ltd.

Tolerance of riverine plants to flooding and exposure indicated by water regime

The flow regime of the River Murray has changed markedly over the last century, and especially the last 50 years, through increased diversions, construction of dams, weirs and levees and changes in operational procedures. A model developed by the Murray–Darling Basin Commission is used to compare simulated natural (unregulated) flows at eight stations with those at seven consecutive stages in the development of regulation. Monthly and annual average flows and coefficients of variation and skewness were computed, and the flow-duration, peak-flow and low-flow characteristics curves plotted. The results confirm that average monthly and annual flows are now considerably lower than those which prevailed under natural conditions. The seasonal distribution of flows has changed in the upper Murray, owing to the influence of dams. Flow-duration characteristics now vary considerably along the river, whereas there was little change under natural conditions. The effect of regulation on flow-duration characteristics is minimal at Albury and becomes more pronounced downstream; it is most apparent in regard to flows exceeded 20–80% of the time. The magnitude of average annual floods (annual exceedance probability 50%) has been reduced by over 50% at all stations, but big floods (average recurrence interval 20 years or more) are little affected. Further, the low flows for a given annual non-exceedance probability are higher under regulated conditions than those under natural conditions. These changes have profound implications for communities of native plants and animals in both riverine and floodplain environments, and also for the long-term utility of the river as a resource.

Effects of regulation on the flow regime of the river Murray, Australia

Before regulation flows in the lower Murray were highly variable, as for most rivers in semi-arid regions. Major floods promoted large-scale recruitment of flora and fauna in riverine and floodplain communities, and seasonal floods maintained lower levels of recruitment. The regime changed with the construction of 10 low-level weirs in 1922-35, supplemented by the effects of dams in upstream areas. Flows remain variable but are much reduced in volume (about 44%). Low flows (100-300 GI per month) have decreased five-fold and moderate flows (500-1500 GI per month) have increased two-fold. Although the magnitude of peak seasonal flows has been diminished, the timing of flows is unaffected. The effects differ in the Valley and Gorge sections of the river, depending on local development of the floodplain and associated wetlands. The weirs have flooded once-temporary wetlands and contributed to problems of salinization. Weir operations cause daily stage fluctuations that diminish downstream, and the channel is developing a stepped gradient as a consequence of active deposition and erosion. Regulation has limited exchanges between the river and its floodplain, changed the nature of the littoral zone and generally created an environment inimical to many native species, notably fish. The key to rehabilitation may be to restore a more natural balance of low and medium flows, but this may be unrealistic. given the needs of irrigators and other water users. Despite its evolutionary history of wide spatial and temporal variation, the Murray river-floodplain ecosystem evidently cannot accommodate these forms of disturbance.

Keith F. Walker

Papers

Flow variability and the ecology of large rivers

A perspective on dryland river ecosystems

Tolerance of riverine plants to flooding and exposure indicated by water regime

Effects of regulation on the flow regime of the river Murray, Australia

Environmental effects of flow regulation on the lower river Murray, Australia