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.

The cell content of 12 bacterial phospholipid fatty acids (PLFA) was determined in bacteria extracted from soil by homogenization/centrifugation. The bacteria were enumerated using acridine orange direct counts. An average of 1.40×10-17 mol bacterial PLFA cell-1 was found in bacteria extracted from 15 soils covering a wide range of pH and organic matter contents. With this factor, the bacterial biomass based on PLFA analyses of whole soil samples was calculated as 1.0–4.8 mg bacterial C g-1 soil C. The corresponding range based on microscopical counts was 0.3–3.0 mg bacterial C g-1 soil C. The recovery of bacteria from the soils using homogenization/centrifugation was 2.6–16% (mean 8.7%) measured by PLFA analysis, and 12–61% (mean 26%) measured as microscopical counts. The soil content of the PLFA 18:2ω6 was correlated with the ergosterol content (r=0.92), which supports the use of this PLFA as an indicator of fungal biomass. The ratio 18:2ω6 to bacterial PLFA is therefore suggested as an index of the fungal:bacterial biomass ratio in soil. An advantage with the method based on PLFA analyses is that the same technique and even the same sample is used to determine both fungi and bacteria. The fungal:bacterial biomass ratio calculated in this way was positively correlated with the organic matter content of the soils (r=0.94).

The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil

Laccases of fungi attract considerable attention due to their possible involvement in the transformation of a wide variety of phenolic compounds including the polymeric lignin and humic substances. So far, more than a 100 enzymes have been purified from fungal cultures and characterized in terms of their biochemical and catalytic properties. Most ligninolytic fungal species produce constitutively at least one laccase isoenzyme and laccases are also dominant among ligninolytic enzymes in the soil environment. The fact that they only require molecular oxygen for catalysis makes them suitable for biotechnological applications for the transformation or immobilization of xenobiotic compounds.

Fungal laccases - occurrence and properties.

Mangroves are woody plants that grow at the interface between land and sea in tropical and sub-tropical latitudes where they exist in conditions of high salinity, extreme tides, strong winds, high temperatures and muddy, anaerobic soils. There may be no other group of plants with such highly developed morphological and physiological adaptations to extreme conditions.

Because of their environment, mangroves are necessarily tolerant of high salt levels and have mechanisms to take up water despite strong osmotic potentials. Some also take up salts, but excrete them through specialized glands in the leaves. Others transfer salts into senescent leaves or store them in the bark or the wood. Still others simply become increasingly conservative in their water use as water salinity increases Morphological specializations include profuse lateral roots that anchor the trees in the loose sediments, exposed aerial roots for gas exchange and viviparous waterdispersed propagules.

Mangroves create unique ecological environments that host rich assemblages of species. The muddy or sandy sediments of the mangal are home to a variety of epibenthic, infaunal, and meiofaunal invertebrates Channels within the mangal support communities of phytoplankton, zooplankton and fish. The mangal may play a special role as nursery habitat for juveniles of fish whose adults occupy other habitats (e.g. coral reefs and seagrass beds).

Because they are surrounded by loose sediments, the submerged mangroves' roots, trunks and branches are islands of habitat that may attract rich epifaunal communities including bacteria, fungi, macroalgae and invertebrates. The aerial roots, trunks, leaves and branches host other groups of organisms. A number of crab species live among the roots, on the trunks or even forage in the canopy. Insects, reptiles, amphibians, birds and mammals thrive in the habitat and contribute to its unique character.

Living at the interface between land and sea, mangroves are well adapted to deal with natural stressors (e.g. temperature, salinity, anoxia, UV). However, because they live close to their tolerance limits, they may be particularly sensitive to disturbances like those created by human activities. Because of their proximity to population centers, mangals have historically been favored sites for sewage disposal. Industrial effluents have contributed to heavy metal contamination in the sediments. Oil from spills and from petroleum production has flowed into many mangals. These insults have had significant negative effects on the mangroves.

Habitat destruction through human encroachment has been the primary cause of mangrove loss. Diversion of freshwater for irrigation and land reclamation has destroyed extensive mangrove forests. In the past several decades, numerous tracts of mangrove have been converted for aquaculture, fundamentally altering the nature of the habitat. Measurements reveal alarming levels of mangrove destruction. Some estimates put global loss rates at one million ha y−1, with mangroves in some regions in danger of complete collapse. Heavy historical exploitation of mangroves has left many remaining habitats severely damaged.

These impacts are likely to continue, and worsen, as human populations expand further into the mangals. In regions where mangrove removal has produced significant environmental problems, efforts are underway to launch mangrove agroforestry and agriculture projects. Mangrove systems require intensive care to save threatened areas. So far, conservation and management efforts lag behind the destruction; there is still much to learn about proper management and sustainable harvesting of mangrove forests.

Mangroves have enormous ecological value. They protect and stabilize coastlines, enrich coastal waters, yield commercial forest products and support coastal fisheries. Mangrove forests are among the world's most productive ecosystems, producing organic carbon well in excess of the ecosystem requirements and contributing significantly to the global carbon cycle. Extracts from mangroves and mangrove-dependent species have proven activity against human, animal and plant pathogens. Mangroves may be further developed as sources of high-value commercial products and fishery resources and as sites for a burgeoning ecotourism industry. Their unique features also make them ideal sites for experimental studies of biodiversity and ecosystem function. Where degraded areas are being revegetated, continued monitoring and thorough assessment must be done to help understand the recovery process. This knowledge will help develop strategies to promote better rehabilitation of degraded mangrove habitats the world over and ensure that these unique ecosystems survive and flourish.

Biology of mangroves and mangrove Ecosystems

The endophytic continuum.

Portions of published procedures for measurement of ergosterol content of decomposing plants were examined for their influence upon ergosterol yield. Common methods of treatment of plant samples prior to sterol extraction (e.g., oven drying, freezing, lyophilization) led to reduced recoveries of ergosterol (ca. 20 to 80%). The least destructive method was direct placement and storage in methanol. Photoconversion of ergosterol is not likely to cause losses during analysis, but losses are likely if there is insufficient mixing during neutral-lipid partitioning from base-hydrolysis reagents. Homogenization (two times for 2 min) and refluxing (2 h) in methanol were equally effective in extracting ergosterol. Direct extraction in base-hydrolysis reagents was less effective (by ca. 40%).

Fundamental procedures for determining ergosterol content of decaying plant material by liquid chromatography.

https://aslopubs.onlinelibrary.wiley.com/doi/pdf/10.4319/lo.1987.32.2.0471

Calculation of cell production of coastal marine bacteria based on measured incorporation of [3H]thymidine1,2

We estimated In si tu grazing rates of heterotrophic nanoplankton (HNAN) on bactenoplankton in a salt marsh estuary using a select~ve metabolic inhibitor technique. Seasonal experiments with prokaryohc inhibitors showed a significant, positive relation between bacterial abundance and HNAN grazing rates. On average, only 40 to 4 5 '/o of bacterioplankton produchon in the estuary appeared to be consumed by phagogrophic Protozoa <20 pm in size. Eukaryotic inhibitor experiments suggested that protozoan grazlng can have a positive feedback effect on bacterioplankton production, and that nitrogen regeneration by Protozoa may be of particular importance to bacterial growth. Although eukaryotic inhibitors did not prove to be rouhnely useful to estimate protozoan grazing rates on bacterioplankton, they may contribute to understanding qualitahve control mechanisms in the microbial food web.

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Trophic interactions between heterotrophic Protozoa and bacterioplankton in estuarine water analyzed with selective metabolic inhibitors

Mutualisms between fungi and fungus-growing animals are model systems for studying coevolution and complex interactions between species. Fungal growing behavior has enabled cultivating animals to rise to major ecological importance, but evolution of farming symbioses is thought to be restricted to three terrestrial insect lineages. Surveys along 2,000 km of North America's Atlantic coast documented that the marine snail Littoraria irrorata grazes fungus-infected wounds on live marsh grass throughout its range. Field experiments demonstrate a facultative, farming mutualism between Littoraria and intertidal fungi. Snails graze live grass primarily not to feed but to prepare substrate for fungal growth and consume invasive fungi. Fungal removal experiments show that snails and fungi act synergistically to suppress marsh grass production. These results provide a case of fungus farming in the marine environment and outside the class Insecta and reveal a previously undemonstrated ecological mechanism (i.e., facilitation of fungal invasion) by which grazers can exert top-down control of marine plant production.

https://www.pnas.org/content/pnas/100/26/15643.full.pdf

Fungal farming in a snail.

This work was supported by NSF grants OCE 82-19866 and OCE 8l-10707 and by grants from the Sapelo Island Research Foundation. Contribution 543 of the University of Georgia Marine Institute.

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Steven Y. Newell

Papers

Fundamental procedures for determining ergosterol content of decaying plant material by liquid chromatography.

Calculation of cell production of coastal marine bacteria based on measured incorporation of [3H]thymidine1,2

Trophic interactions between heterotrophic Protozoa and bacterioplankton in estuarine water analyzed with selective metabolic inhibitors

Fungal farming in a snail.

Small, aloricate ciliates as a major component of the marine heterotrophic nanoplankton1