Enzymatic profiles of 11 barophilic bacteria under in situ conditions: evidence for pressure modulation of phenotype.

Barophilic bacteria are microorganisms that grow preferentially (facultative barophiles) or exclusively (obligate barophiles) under elevated hydrostatic pressure. Barophilic bacteria have been isolated from a variety of deep-sea environments. Attempts to characterize these organisms have been hampered by a lack of appropriate methodologies. A colorimetric method for the detection of 19 constitutively expressed enzymes under in situ conditions of pressure and temperature has been devised, using a simple modification of the commercially available API ZYME enzyme assay kit. By using this method, enzyme profiles of 11 barophilic isolates, including an obligate barophile, were determined. Nine of the 10 facultatively barophilic isolates examined exhibited a change of phenotype in at least one enzyme reaction when tested at 1 atm (1 atm = 101.29 kPa), compared with results obtained under in situ pressure. The assay is simple and rapid and allows for direct determination of enzyme activity under conditions of high pressure and low temperature.     

Enzymatic profiles of 11 barophilic bacteria under in situ conditions: evidence for pressure modulation of phenotype

Barophilic bacteria are microorganisms that grow preferentially (facultative barophiles) or exclusively (obligate barophiles) under elevated hydrostatic pressure. Barophilic bacteria have been isolated from a variety of deep-sea environments. Attempts to characterize these organisms have been hampered by a lack of appropriate methodologies. A colorimetric method for the detection of 19 constitutively expressed enzymes under in situ conditions of pressure and temperature has been devised, using a simple modification of the commercially available API ZYME enzyme assay kit. By using this method, enzyme profiles of 11 barophilic isolates, including an obligate barophile, were determined. Nine of the 10 facultatively barophilic isolates examined exhibited a change of phenotype in at least one enzyme reaction when tested at 1 atm (1 atm = 101.29 kPa), compared with results obtained under in situ pressure. The assay is simple and rapid and allows for direct determination of enzyme activity under conditions of high pressure and low temperature.     

Properties of the Glucose Transport System in Some Deep-Sea Bacteria

Many deep-sea bacteria are specifically adapted to flourish under the high hydrostatic pressures which exist in their natural environment. For better understanding of the physiology and biochemistry of these microorganisms, properties of the glucose transport systems in two barophilic isolates (PE-36, CNPT-3) and one psychrophilic marine bacterium (Vibrio marinus MP1) were studied. These bacteria use a phosphoenol-pyruvate:sugar phosphotransferase system (PTS) for glucose transport, similar to that found in many members of the Vibrionaceae and Enterobacteriaceae. The system was highly specific for glucose and its nonmetabolizable analog, methyl alpha-glucoside (a-MG), and exhibited little affinity for other sugars tested. The temperature optimum for glucose phosphorylation in vitro was approximately 20°C. Membrane-bound PTS components of deep-sea bacteria were capable of enzymatically cross-reacting with the soluble PTS enzymes of Salmonella typhimurium, indicating functional similarities between the PTS systems of these organisms. In CNPT-3 and V. marinus, increased pressure had an inhibitory effect on a-MG uptake, to the greatest extent in V. marinus. Relative to atmospheric pressure, increased pressure stimulated sugar uptake in the barophilic isolate PE-36 considerably. Increased hydrostatic pressure inhibited in vitro phosphoenolpyruvate-dependent a-MG phosphorylation catalyzed by crude extracts of V. marinus and PE-36 but enhanced this activity in crude extracts of the barophile CNPT-3. Both of the pressure-adapted barophilic bacteria were capable of a-MG uptake at higher pressures than was the nonbarophilic psychrophile, V. marinus.     

“Micronucleoli” in theXenopus germinal vesicle

The germinal vesicle of the Xenopus oocyte contains 1500 or more extrachromosomal nucleoli that are assembled on amplified copies of the rRNA genes. Many of these nucleoli have diameters of 10–15 μm, but some are much smaller, ranging down to 1 μm or less. Morphologically the smaller nucleoli or ”micronucleoli” resemble the similarly sized B snurposomes, but they can be recognized with appropriate antibody probes (e.g., anti-nucleolin and anti-fibrillarin). We describe here a sensitive fluorescent staining technique that uses avidin and propidium iodide to visualize the rDNA in the amplified nucleoli. Many large nucleoli stain about as brightly as haploid yeast nuclei on the same slides. They presumably contain about 12 Mb of DNA, equivalent to 900 rDNA repeats. The smallest micronucleoli display only a tiny dot of stain, which must correspond to relatively few rDNA repeats. Received: 8 January 1997; in revised form: 20 January 1997 / Accepted: 27 January 1997     

Pressure and temperature effects on growth and viability of the hyperthermophilic archaeon Thermococcus peptonophilus

We studied the effects of high temperatures and elevated hydrostatic pressures on the physiological behavior and viability of the extremely thermophilic deep-sea archaeon Thermococcus peptonophilus. Maximal growth rates were observed at 30 and 45 MPa although no significant increases in cell yields were detected. Growth at 60 MPa was slower. The optimal growth temperature shifted from 85° C at 30 MPa to 90–95° C at 45 MPa. Cell viability during the stationary phase was also enhanced under high pressure. A trend towards barophily at pressures greater than those encountered in situ at the sea floor was demonstrated at increasing growth temperatures. The viability of cells during starvation, at high temperature (90, 95° C), and at low temperature (10° C) was enhanced at 30 and 45 MPa as compared to atmospheric pressure. These results show that the extremely thermophilic archaeon T. peptonophilus is a barophile. Received: 21 October 1996 / Accepted: 5 February 1997     

Purification of a ccb-type quinol oxidase specifically induced in a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F

We investigated for the first time the respiratory chain system of a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F. A membrane-bound ccb-type quinol oxidase, from cells grown at 60 MPa pressure, was purified to an electrophoretically homogeneous state. The purified enzyme complex consisted of four kinds of subunits with molecular masses of 98, 66, 18.5, and 15 kDa, and it contained 0.96 mol of protoheme and 1.95 mol of covalently bound heme c per mol of enzyme. Only protoheme in the enzyme reacted with CO and CN⁻, and the catalytic activity of the enzyme was 50% inhibited by 4 μM CN⁻. The isoelectric point of the native enzyme complex was determined to be 5.0. This enzyme was specifically induced only under conditions of elevated hydrostatic pressure, and high levels were expressed in cells grown at 60 MPa. The membranes isolated from cells grown at atmospheric pressure (0.1 MPa) exhibited high levels of both cytochrome c oxidase and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPDH2)-oxidase activity. These results suggest the presence of two kinds of respiratory chains regulated in response to pressure in the deep-sea bacterium DB-172F. Received: November 25, 1997 / Accepted: December 25, 1997     

Shoot dynamics and architecture of saplings in Fagus crenata across its geographical range

 Relationships between leaf or shoot size, number, and arrangement in response to light were investigated to test the hypothesis that these characteristics are linked. In order to test this hypothesis, the divergence in allometry and shoot dynamics in saplings of Japanese beech (Fagus crenata) obtained from four populations and having different leaf sizes were examined in a nursery under both full sun and shade conditions. Trees with different leaf sizes also showed large differences in canopy structure, particularly when shade-grown saplings were compared. The final leaf mass distributions of the large-leaf populations were conical or “bottom - heavy”, while those of the small-leaf populations were planar or “top - heavy”. The slope of the allometric relations between leaf mass and shoot and branch mass in small-leaved populations were steeper than those in large-leaf populations. The four populations were classified into two growth types: populations producing a few large leaf and shoot modules corresponded to “stem growth type”, and those producing many small leaf and shoot modules corresponded to “leaf growth type”. These kinds of intra-specific variation in architecture and growth of F. crenata trees may influence the structure and dynamics of forests in accordance with differences in competitive ability or sensitivity to disturbances such as windstorm. Received: 18 March 1997 / Accepted: 21 October 1997     

Revision of the genus <Emphasis Type="Italic">Nuchequula</Emphasis> with descriptions of three new species (Perciformes: Leiognathidae)

The leiognathid genus Nuchequula can be defined by the following combination of characters: mouth protruding downward; a narrow band of small, slender, villiform teeth in both jaws; teeth on upper jaw strongly recurved; the lateral line almost complete; a dark blotch on the nape. Although the genus was first established as a subgenus of Eubleekeria, it is here raised to generic level on the basis of the aforementioned morphological characters and recent molecular biological evidence. The genus comprises six valid species: N. blochii (Valenciennes 1835), distributed in India and Thailand; N. flavaxilla sp. nov., occurring only at Panay I., Philippines; N. gerreoides (Bleeker 1851), widely distributed in the Indo-West Pacific, from the Persian Gulf to Cape York, Australia, and north to Taiwan; N. glenysae sp. nov., from northern Australia and Ambon, Indonesia; N. longicornis sp. nov., from the Gulf of Thailand and Indonesia; and N. nuchalis (Temminck and Schlegel 1845), occurring in southern China including Taiwan, and southern Japan. Diagnostic characters of the species belonging to the genus are as follows: N. blochii—breast scaled, cheek naked, and a conspicuous black blotch distally on spinous dorsal fin; N. flavaxilla sp. nov.—breast naked, dorsolateral body surface fully scaled, preorbital spine bicuspid and not expanded distally, and second dorsal and anal fin spines conspicuously elongated; N. gerreoides—breast naked, anterior part of dorsolateral surface of body almost completely scaled, and second dorsal and anal fin spines not conspicuously elongated; N. glenysae sp. nov.—breast completely scaled, cheek scaled, and unique complicated sensory canals present on the suborbital area, extending to the nape; N. longicornis sp. nov.—breast naked, dorsolateral body surface fully scaled, preorbital spine bicuspid or tricuspid and extended distally, and second dorsal fin spines only conspicuously elongated; N. nuchalis—breast naked, anterior part of dorsolateral surface of body widely naked, and a conspicuous dark blotch distally on spinous dorsal fin.     

A reexamination of Gilligan’s analysis of the female moral system

Gilligan’s (1982) refinement of Kohlberg’s theory on moral development operates on two theses: (1) females, more so than males, reach moral decisions based on the personalities of the relevant individuals; and (2) female behaviors stemming from moral decisions are based upon “care” and “responsibility for others.” This article accepts the first thesis but argues that the second is incorrect. That is, self-interest—i.e., aiding “blood” kin and/or carefully monitoring reciprocity—rather than “altruism” is argued to be the operant dynamic in forging distaff morality and resultant behavior. Six empirical examples are presented as contraindicative of Gilligan’s second thesis. Finally, it is suggested that selection for the psychological traits of independence and the mastery of subtle social chess yielded ancestral females who had more descendants—us—than did females with alternative profiles. Nancy S. Coney is a professor at Western Illinois University and is interested in both clinical and nonclinical aspects of women’s psychology. Wade C. Mackey is interested in biocultural anthropology and has authored two books on fathering:Fathering Behaviors (Plenum, 1995) andThe American Father (Plenum, 1996).     

Analysis of complex leaf and flower characters in Rhododendron using a molecular linkage map

 A molecular linkage map of Rhododendron has been constructed by using a segregating population from an interspecific cross. Parent-specific maps based on 239 RAPD, 38 RFLP, and two microsatellite markers were aligned using markers heterozygous in both parents. The map of the male parent ‘Cunningham’s White’ comprised 182 DNA markers in 13 linkage groups corresponding to the basic chromosome number. In the female parent ‘Rh 16’ 168 markers were located on 18 linkage groups. An assignment of putative homologous linkage groups was possible for 11 groups of each parent. QTL analyses based on the non-parametric Kruskal-Wallis rank-sum test were performed for the characters “leaf chlorosis” and “flower colour” scored as quantitative traits. For leaf chlorosis, two genomic regions bearing QTLs with significant effects on the trait were identified on two linkage groups of the chlorosis-tolerant parent. RAPD marker analysis of additional lime-stressed genotypes tested under altered environmental conditions verified the relationship between marker allele frequencies and the expression of chlorosis. Highly significant QTL effects for flower colour were found on two chromosomes indicating major genes located in these genome areas. The prospects for utilization of a linkage map in Rhododendron are discussed. Received: 28 September 1998 / Accepted: 5 November 1998     

Analysis of changes in congener selectivity during PCB degradation by Burkholderia sp. strain TSN101 with increasing concentrations of PCB and characterization of the bphBCD genes and gene products

We isolated and characterized a gram-negative bacterium, Burkholderia sp. strain TSN101, that can degrade polychlorinated biphenyls (PCBs) at concentrations as high as 150 μg Kaneclor 300/ml, a PCB mixture equivalent to Aroclor 1242. Growing cells of strain TSN101 degraded most of the tri- and tetrachlorobiphenyls in medium containing 25 μg Kaneclor 300/ml. Using PCB concentrations of 50–150 μg of Kaneclor 300/ml, the congener selectivity pattern was different and the pattern of chlorine substitution strongly affected degradation of some congeners. At 25 μg Kaneclor 300/ml, strain TSN101 degraded di- and trichlorinated congeners with chlorine substitutions at both the ortho and the para positions. At higher concentrations of Kaneclor 300, di- and trichlorobiphenyls with ortho substituents in both phenyl rings were not degraded well. Trichlorobiphenyls with para and meta substitutents were degraded equally well at all concentrations studied. The ability of strain TSN101 to degrade ortho and para-substituted congeners was confirmed using a defined PCB mixture with chlorine substituents at 2′- and 4′-positions. A 5-kb DNA fragment containing the bphBCD genes was cloned and sequenced. Comparison of the deduced amino acid sequences of these genes with related proteins indicated 99 and 98% sequence similarity to the BphB and BphD of Comamonas testosteroni strain B-356, respectively. The bphC gene product showed 74% sequence similarity to the BphC of Burkholderia cepacia strain LB400 and exhibited a narrow substrate specificity with strong affinity for 2,3-dihydroxybiphenyl. A bphC-disrupted mutant of Burkholderia sp. strain TSN101, constructed by gene replacement, lost the ability to utilize biphenyl, thus supporting the role of the cloned bph gene in biphenyl metabolism. Received: 18 February 1997 / Accepted: 19 August 1997     

Genetic analysis of the dwarfing gene Rht8 in wheat. Part II. The distribution and adaptive significance of allelic variants at the Rht8 locus of wheat as revealed by microsatellite screening

 Wheat microsatellite WMS 261 whose 192-bp allele has been shown to be diagnostic for the commercially important dwarfing gene Rht8 was used to screen over 100 wheat varieties to determine the worldwide spread of Rht8. The results showed Rht8 to be widespread in southern European wheats and to be present in many central European wheats including the Russian varieties ‘Avrora’, ‘Bezostaya’ and ‘Kavkaz’. Rht8 appears to be of importance to South European wheats as alternative giberellic acid (GA)-insensitive dwarfing genes do not appear to be adapted to this environment. The very successful semi-dwarf varieties bred by CIMMYT, Mexico, for distribution worldwide have been thought to carry Rht8 combined with GA-insensitive dwarfing genes. Additional height reduction would have been obtained from pleiotropic effects of the photoperiod-response gene Ppd1 that is essential to the adaptability of varieties bred for growing under short-winter days in tropical and sub-tropical areas. The microsatellite analysis showed that CIMMYT wheats lack Rht8 and carry a WMS 261 allelic variant of 165 bp that has been associated with promoting height. This presumably has adaptive significance in partly counteracting the effects of other dwarfing genes and preventing the plants being too short. Most UK, German and French wheats carry an allelic variant at the WMS 261 locus with 174 bp. This could be selected because of linkage with the recessive photoperiod-sensitive ppd1 allele that is thought to offer adaptive significance northern European wheats. Received: 17 October 1997 / Accepted: 12 November 1997     

Directed formation of chromosomal deletions in Salmonella typhimurium : targeting of specific genes induced during infection

In vivo expression technology (IVET) has resulted in the isolation of more than 100 Salmonella typhimurium genes that are induced during infection. Many of these in vivo induced (ivi) genes, as well as other virulence genes, are clustered in regions of the chromosome that are specific for Salmonella and are not present in Escherichia coli (e.g., pathogenicity islands). It would be desirable to be able to delete such putative virulence regions of the chromosome, and if the deletion removes genes that play a role in pathogenesis subsequent efforts can then be focused on individual genes that reside within that region. We therefore have developed a strategy for constructing chromosomal deletions which are not limited in size, have defined endpoints with a selectable marker at the joint point, and are not dependent on prior knowledge of sequences contained within the deleted region. Such deletion strategies can be applied to almost any bacterium with homologous recombination and to plasmid-based mutational systems where homologous recombination is not desired or feasible. Received: 6 October 1997 / Accepted: 30 December 1997     

Morphology,ontogeny, and heterochrony in lower and middle Cambrian Gogiids (Eocrinoidea,Echinodermata) from Guizhou Province,China

Gogiid eocrinoids from the Lower Cambrian (Balang Formation — Guizhoueocrinus) and basal Middle Cambrian (Kaili Formation — Sinoeocrinus, Globoeocrinus) in Guizhou Province, China are found in great numbers and are extremely well preserved as high fidelity molds in shale/mud sized siliciclastics. Because of their numbers, complete ontogenetic growth sequences have been observed. Significant differences in growth patterns (heterochrony) are present between Lower and Middle Cambrian genera: thecal plates tend to be paedomorphic in development, while the sutural pores developed between them are peramorphic in their development. Because of the large surface area of theca and brachioles relative to the size of the attachment area, considerable drag in ambient currents would necessitate a strong attachment medium. Anchoring by “biogluing”, possibly by collagen, directly to the substrate or to biodetritus is proposed as the method of attachment.     

Suppressors of the temperature sensitivity of DNA polymerase α mutations in Saccharomyces cerevisiae

We have isolated two high copy, allele-specific suppressors of the temperature sensitivity of mutations in POL1, the gene that encodes the catalytic subunit of DNA polymerase α in the yeast Saccharomyces cerevisiae. Both genes, PSP1 and PSP2, also partially suppressed a mutation in POL3 which encodes DNA polymerase δ, and both also affected a mutation in CDC6, which acts in initiation of DNA replication. Suppression was not general, since ts mutations in several genes unrelated to replication were not affected. PSP1 was partially effective on low-copy-number vectors, while PSP2 required high copy numbers. The presence of suppressing plasmids did not alter the steady-state level of Pol1 protein, so suppression does not appear to be due to an increase in production or stability of Pol1p. Deletion of either PSP gene or both in combination resulted in apparently normal viable cells. While neither gene is homologous to genes with known functions, PSP1 and PSP2 both have unusual amino acid compositions: PSP1 is rich in asparagine and glutamine, while PSP2 is rich in asparagine and contains “RGG” motifs that have been associated with RNA-binding proteins. We also describe a transposon-mediated strategy that should be generally effective for rapid characterization of multicopy suppressors. Received: 20 July 1997 / Accepted: 1 October 1997     

Competitive reversals inside ecological reserves: the role of external habitat degradation

 Habitat degradation is the slow – and often subtle – deterioration in habitat quality that accompanies human activities through increases in road density, pesticide use, hunting pressure, etc. Such degradation is of particular concern in fragmented habitats where economic or jurisdictional boundaries rather than ecological ones determine the level of exploitation adjoining habitat patches endure. To examine the consequences habitat degradation might have on species interactions, we posited a patch of pristine habitat surrounded by “matrix” habitat whose degradation level was variable. Using a coupled pair of diffusive Lotka–Volterra competition equations with Robin (mixed) boundary conditions, we modeled the dynamics of two competing species inhabiting the pristine patch and incorporated matrix degradation through a tunable “hostility” parameter representing species’ mortality rates in the matrix. We found that the numerical range of competition coefficients over which one species is the competitive dominant and the other inferior may grow or shrink as matrix quality deteriorates. In some cases, degradation of the exterior habitat would bring about a complete competitive reversal inside the preserve. This result, wherein a formerly inferior species supplants a formerly dominant one – even inside the “protected” remnant patch itself – has policy implications for both nature reserve design and management of human activities outside park boundaries. Received: 30 April 1997     

Taxonomic studies of deep-sea barophilic Shewanella strains and description of Shewanella violacea sp. nov.

Several barophilic Shewanella species have been isolated from deep-sea sediments at depths of 2,485– 6,499 m. From the results of taxonomic studies, all of these isolates have been identified as strains of Shewanella benthica except for strain DSS12. Strain DSS12 is a member of a novel, moderately barophilic Shewanella species isolated from the Ryukyu Trench at a depth of 5,110 m. On Marine Agar 2216 plates, this organism produced a violet pigment, whereas the colonies of other isolates (S. benthica) were rose-colored. Phylogenetic analysis based on 16 S ribosomal RNA gene sequences showed that strain DSS12 represents a separate lineage within the genus Shewanella that is closely related to S. benthica and particularly to the members of the Shewanella barophiles branch. The temperature range for growth and some of the biochemical characteristics indicate that strain DSS12 differs from other Shewanella species. Furthermore, strain DSS12 displayed a low level of DNA similarity to the Shewanella type strains. Based on these differences, it is proposed that strain DSS12 represents a new deep-sea Shewanella species. The name Shewanella violacea (JCM 10179) is proposed. Received: 15 May 1998 / Accepted: 15 July 1998     

Subtropical coral-reef associated sedimentary facies characterized by molluscs (Northern Bay of Safaga, Red Sea, Egypt)

Summary The shallow marine subtropical Northern Bay of Safaga is composed of a complex pattern of sedimentary facies that are generally rich in molluscs. Thirteen divertaken bulk-samples from various sites (reef slopes, sand between coral patches, muddy sand, mud, sandy seagrass, muddy seagrass, mangrove channel) at water depths ranging from shallow subtidal to 40m were investigated with regard to their mollusc fauna >1mm, which was separated into fragments and whole individuals. Fragments make up more than 88% of the total mollusc remains of the samples, and their proportions correspond to characteristics of the sedimentary facies. The whole individuals were differentiated into 622 taxa. The most common taxon,Rissoina cerithiiformis, represented more than 5% of the total mollusc content in the samples. The main part of the fauna consists of micromolluscs, including both small adults and juveniles. Based on the results of cluster-, correspondence-, and factor analyses the fauna was grouped into several associations, each characterizing a sedimentary facies: (1) “Rhinoclavis sordidula—Corbula erythraeensis-Pseudominolia nedyma association” characterizes mud. (2) “Microcirce sp.—Leptomyaria sp. association” characterizes muddy sand. (3)”Smaragdia spp.-Perrinia stellata—Anachis exilis—assemblage” characterizes sandy seagrass. (4) “Crenella striatissima—Rastafaria calypso—Cardiates-assemblage” characterizes muddy seagrass. (5) “Glycymeris spp.-Parvicardium sueziensis-Diala spp.-assemblage” characterizes sand between coral patches. (6) “Rissoina spp.-Triphoridae —Ostreoidea-assemblage” characterizes reef slopes. (7) “Potamides conicus—Siphonaria sp. 2—assemblage” characterizes the mangrove. The seagrass fauna is related to those of sand between coral patches and reef slopes with respect to gastropod assemblages, numbers of taxa and diversity indices, and to the muddy sand fauna on the basis of bivalve assemblages and feeding strategies of bivalves. The mangrove assemblage is related to those of sand between coral patches and the reef slope with respect to taxonomic composition and feeding strategies of bivalves, but has a strong relationship to those of the fine-grained sediments when considering diversity indices. Reef slope assemblages are closely related to that of sand between coral patches in all respects, except life habits of bivalves, which distincly separates the reef slope facies from all others.     

The Quantum Co-Evolution Unit: An Example of ‘Awa (Kava—<Emphasis Type="Italic">Piper methysticum</Emphasis> G. Foster) in Hawaiian Culture

The Quantum Co-Evolution Unit: An Example of ‘Awa (Kava— Piper methysticum G. Foster) in Hawaiian Culture. The process of co-evolution occurs in many kinds of relationships and on various scales. One example of a co-evolutionary relationship is that of a plant and a culture with which it interacts. Such relationships are dynamic and ever changing. Researchers have discussed this concept and its implications for decades, yet no quantifiable unit or standardized scale has been accepted with which to measure this change. The theoretical “quantum co-evolution unit” (QCU) is proposed as the smallest measurable scale of interactions between plants and people. A collection of QCUs for a linked plant and human population would be its “ethnobotanical population.” This could be measured at various points in time to quantify the changing relationships between plants and people. These models set up a structure to discuss methodologies for quantifying co-evolutionary relationships such as are seen in the evolution of ethnobotanical populations. The co-evolving relationship between ‘awa (kava—Piper methysticum) and Hawaiian culture is used as an example to illustrate this idea.