Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium

Dundas, Ian E. D. (University of Illinois, Urbana), and H. Orin Halvorson. Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium. J. Bacteriol. 91:113-119. 1966.-Arginine was shown to be essential for growth of Halobacterium salinarium strain 1 in a chemically defined medium. Citrulline was the only compound which could substitute for arginine without affecting growth. Resting cells of H. salinarium converted arginine to citrulline and citrulline to ornithine. Cells grown in an arginine-free medium with C(14)-ureido-labeled citrulline incorporated the isotope mainly into the arginine of their proteins. The enzymes arginine desimidase and ornithine transcarbamylase were found and studied in cell-free extracts of H. salinarium. Experiments indicated that arginine was degraded in H. salinarium by arginine desimidase to citrulline, and that citrulline was further degraded by ornithine transcarbamylase to carbamyl phosphate and ornithine. Synthesis of arginine from citrulline seems to occur via the formation of argininosuccinic acid.     

Ketocarotenoids in halobacteria: 3-hydroxy-echinenone and trans-astaxanthin

HPLC testing of the carotenoid content of the halobacteria Halobacterium salinarium, Haloarcula hispanica and Haloferax mediterranei showed that all contained high amounts of ketocarotenoids. Halobacterium salinarium produced 2400 μg of total carotenoids per gram of dried bacteria, including 265 μg of trans -astaxanthin (11%), and 588 μg of 3-hydroxy-echinenone (24%). The biotechnological properties of Halobacterium salinarium as a natural pigment source are also presented. The results are compared with those from the yeast Phaffia rhodozyma currently used in the industry as a source of trans -astaxanthin.     

Survival and sublethal responses of early life stages of pike exposed to low pH in artificial post-mining lake water

The threshold pH for survival of early life stages of pike Esox lucius in post-mining lakes was determined in a laboratory experiment using artificial water characterized by high concentrations of dissolved Ca, Al, Fe, Mn and SO2/4⁻. At pH 3·50–4·00, high mortality was observed before hatching. At pH 4·00 and 4·25, hatching rates were reduced compared with the controls (pH 7·40), and many embryos died in a partly hatched state. Hatching success of embryos exposed to pH levels of 4·50 or higher was not affected. However, at pH 5·00 many newly hatched embryos were deformed. Furthermore, pike exposed to pH 4·00–5·00 did not start feeding. At pH 4·75 and below, mortality increased to 100%, and at pH 5·00, only few eleutheroembryos which were in extremely poor condition survived to the end of the experiment. At pH 5·50, survival was in the same range as in the control group, but growth was reduced. Therefore, early life stages of pike are expected to survive in Lustian post-mining lakes when a pH of 5·50 and above is reached and maintained.     

Ontogeny of the gastrointestinal tract and selected digestive enzymes in cobia Rachycentron canadum (L.)

The ontogeny of the digestive system of cobia Rachycentron canadum from hatching to 22 days post-hatch (dph) (20·1 mm standard length) was examined with light microscopy. The activities of selected pancreatic enzymes were also determined during this period in order to optimize current rearing methods for this species. At hatching (3·6 mm), the digestive tract consisted of a relatively undifferentiated, straight tube positioned dorsally to the yolk sac. The major morphological changes in the digestive tract primarily occurred over the first 1–4 dph (3·6–4·4 mm). During this time, larvae began exogenous feeding (3 dph) and the digestive tract differentiated into five histologically distinct regions: buccopharynx, oesophagus, stomach anlage, anterior intestine and posterior intestine. Yolk reserves were exhausted by 5 dph (4·5 mm) and the oil globule began rapidly decreasing in size disappearing entirely by 9–10 dph (6·3–6·8 mm). Gastric glands differentiated at this time, and by 12 dph (8·1 mm) surface mucous cells of the stomach anlage stained positive for neutral mucosubstances. By 16 dph (11·6 mm), the blind sac (fundic region) of the stomach formed as did the pyloric caecae which initially appeared as a single protrusion of the anterior intestine just ventral to the pyloric sphincter. Generally, enzyme activities (U larva⁻¹) for amylase (0·0–1·8), chymotrypsin (0·0–7902·4), trypsin (0·2–16·6) and lipase (9·3–1319·0) were measurable at or soon after hatching and increased steadily from c. 8–22 dph (5·7–20·1 mm). The results of this study are discussed in terms of current and future weaning practices of this species.     

The effects of environmental factors on rainbow smelt Osmerus mordax embryos and larvae

Experiments were conducted to identify environmental factors that influence the survival of rainbow smelt Osmerus mordax during their early life stages. Developing rainbow smelt embryos and yolk-sac larvae were cultured under controlled conditions with different dissolved oxygen (DO; 1·09, 2·18, 4·37 and 6·55 mg l⁻¹, pH (4·0, 4·5, 5·0, 5·5, 6·0 and 7·0), nitrate ( 0·7, 3·6, 7·3, 14·6 and 29·2 mg l⁻¹), phosphate (0·04, 0·21, 0·42, 2·08 and 4·17 mg l⁻¹) and salinity (0, 5, 10, 15, 20 and 30) levels. Rainbow smelt embryos were also incubated with simulated tidal salinity fluctuations (2–28), ultraviolet radiation (irradiances of 2·8, 6·2 and 5·1 W m⁻²) and under natural conditions in two rainbow smelt spawning rivers. In the laboratory, hatch was only impaired under the lowest DO and pH conditions (0 and 13% hatch, respectively) and at highest constant salinity levels (0% hatch). Larval survival was only affected by pH levels ≤5·0. The experiment that compared hatch under natural conditions was terminated when embryos became covered with silt and fungus. These results suggest that water acidification, sediment and fungal growth may affect rainbow smelt survival during their early life stages.     

Osteological development of the vertebral column and of the caudal complex in Dentex dentex

Osteological development of the vertebral column and caudal complex in common dentex was described under extensive larval rearing conditions. Generally, the cartilaginous bones developed prior to the membranous bones. The development of the axial skeleton began with the formation of the hypural 1, the neural arches 2 and 3, as well as the haemal arches 1–8 at 4·8, 4·9 and 5·0 mm total length ( L _T, measured in vivo ), respectively. By 7·5 mm L _T, all the cartilaginous elements were formed, except for the ventral ribs, which formed between the range of 8·4–18·0 mm L _T. The caudal lepidotrichia were the first membranous bones to appear (5·3 mm L _T) and attain their full meristic count (7·4 mm L _T), followed by the vertebral centra, which formed between 6·6 and 9·7 mm L _T. By 25·0 mm L _T, all the elements were fully ossified except for the ventral ribs. The developmental direction and order of all the elements were studied with respect to their formation and ossification. The results were discussed in the contexts of ichthyoplankton, ecology and aquaculture. Compared with other Sparidae species, common dentex followed a pattern of relatively rapid rate of osteological development.     

Composition, structure and distribution of the ichthyoplankton in a Mediterranean coastal lagoon

The species composition and assemblage structure of the ichthyoplankton from the Mar Menor Lagoon in south‐east Spain are given. The fish larvae were sorted from zooplankton samples collected at 20 stations with a plankton net (50 cm mouth diameter and 500 μm mesh) during 36 surveys between February and December 1997. A total of 39 575 fish larvae representing 14 families, 22 genera and 36 species were identified. Gobiidae was the most dominant family (77·0%) followed by Blenniidae (19·4%) and Atherinidae (1·3%). The most abundant species were Gobius niger and Gobius paganellus , which accounted for 42·7 and 19·3% of the total respectively. These species were followed in order of relative abundance by Pomatoschistus marmoratus (13·9%), Parablennius pilicornis (9·4%), Lipophrys pavo (7·7%), Atherina boyeri (1·3%) and Parablennius tentacularis (1·3%). The high species diversity (2·0–2·8 bits individual⁻¹ for the annual diversity spectra at each sampling station) reflected a diverse assemblage of species. The main commercial species in the lagoon (Sparidae and Mugilidae) were poorly represented among the ichthyoplankton and they probably enter the lagoon on the bottom as recruits. Chlorophyll a concentration in the water column was the main factor explaining the seasonal variation in larval abundance. Spatial distribution of larvae was related to hydrographical circulation patterns in the lagoon and the movement of marine‐spawned larvae through the channels connecting the lagoon with the Mediterranean Sea.     

Thermal tolerance of a northern population of striped bass Morone saxatilis

Thermal tolerance of age 0+ year Shubenacadie River (Nova Scotia, Canada) striped bass Morone saxatilis juveniles (mean ± s . e . fork length, L _F, 19·2 ± 0·2 cm) acclimated in fresh water to six temperatures from 5 to 30° C was measured by both the incipient lethal technique (72 h assay), and the critical thermal method ( C _m). The lower incipient lethal temperature ranged from 2·4 to 11·3° C, and the upper incipient lethal temperature ( I _U) from 24·4 to 33·9° C. The area of thermal tolerance was 618° C². In a separate experiment, the I _U of large age 2+ year fish (34·4 ± 0·5 cm L _F) was 1·2 and 0·6° C lower ( P < 0·01) than smaller age 1+ year fish (21·8 ± 0·5 cm L _F) at acclimation temperatures of 16 and 23° C. Using the C _m, loss of equilibrium occurred at 27·4–37·7° C, loss of righting response at 28·1–38·4° C and onset of spasms at 28·5–38·8° C, depending on acclimation temperature. The linear regression slopes for these three responses were statistically similar (0·41; P > 0·05), but the intercepts differed (25·3, 26·0 and 26·5° C; P < 0·01). The thermal tolerance of this northern population appears to be broader than southern populations.     

Formation of insoluble magnesium phosphates during growth of the archaea Halorubrum distributum and Halobacterium salinarium and the bacterium Brevibacterium antiquum

Stationary phase cells of the halophilic archaea Halobacterium salinarium and Halorubrum distributum, growing at 3-4 M NaCl, and of the halotolerant bacterium Brevibacterium antiquum, growing with and without 2.6 NaCl, took up approximately 90% of the phosphate from the culture media containing 2.3 and 11.5 mM phosphate. The uptake was blocked by the uncoupler FCCP. In B. antiquum, EDTA inhibited the phosphate uptake. The content of polyphosphates in the cells was significantly lower than the content of orthophosphate. At a high phosphate concentration, up to 80% of the phosphate taken up from the culture medium was accumulated as Mg(2)PO(4)OH x 4H(2)O in H. salinarium and H. distributum and as NH(4)MgPO(4) x 6H(2)O in B. antiquum. Consolidation of the cytoplasm and enlargement of the nucleoid zone were observed in the cells during phosphate accumulation. At phosphate surplus, part of the H. salinarium and H. distributum cell population was lysed. The cells of B. antiquum were not lysed and phosphate crystals were observed in the cytoplasm.     

Survival of Escherichia coli O157 : H7 in yoghurt during preparation and storage at 4°C

Cow's milk was inoculated with ca 10³ and 10⁷ cfu ml⁻¹ Escherichia coli O157 : H7. After fermentation at 42°C for 0–5 h, the yoghurt was stored at 4°C. Two kinds of yoghurt were used : traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E. coli O157 : H7 decreased from 3·52 to 2·72 log₁₀ cfu ml⁻¹ and from 7·08 to 5·32 log₁₀ cfu ml⁻¹ in TY, and from 3·49 to 2·73 log₁₀ cfu ml⁻¹ and from 7·38 to 5·41 log₁₀ cfu ml⁻¹ in BY. The pH values of yoghurt dropped from 6·6 to 4·5 and 4·4 in TY (for low and high pathogen inocula, respectively), and from 6·6 to 4·6 and 4·5 in BY (for low and high pathogen inocula, respectively).     

Identification and tracing of Enterococcus spp. by RAPD-PCR in traditional fermented sausages and meat environment

Aims:  Four local small-scale factories were studied to determine the sources of enterococci in traditional fermented sausages.
Methods and Results:  Different points during the production of a traditional fermented sausage type ( fuet ) were evaluated. Randomly amplified polymorphic DNA (RAPD)-PCR was used to type 596 Enterococcus isolates from the final products, the initial meat batter, the casing, the workers' hands and the equipment. Species-specific PCR-multiplex and the partial sequencing of atpA gene and 16S rRNA gene sequencing allowed the identification of the isolates: Enterococcus faecalis (31·4%), Enterococcus faecium (30·7%), Enterococcus sanguinicola (14·9%), Enterococcus devriesei (9·7%), Enterococcus malodoratus (7·2%), Enterococcus gilvus (1·0%), Enterococcus gallinarum (1·3%), Enterococcus casseliflavus (3·4%), Enterococcus hermanniensis (0·2%), and Enterococcus durans (0·2%) . A total of 92 different RAPD-PCR profiles were distributed among the different factories and samples evaluated. Most of the genotypes found in fuet samples were traced back to their source.
Conclusions:  The major sources of enterococci in the traditional fermented sausages studied were mainly the equipment followed by the raw ingredients, although a low proportion was traced back to human origin.
Significance and Impact of the Study:  This work contributes to determine the source of enterococcal contamination in fermented sausages and also to the knowledge of the meat environment.     

Interannual and regional variations in body length, weight and energy content of age-0 Pacific herring from Prince William Sound, Alaska

Age-0 Pacific herring were surveyed in October of 4 years in a large northern Gulf of Alaska estuary, to determine the range of variations in length, weight and whole body energy content (WBEC). These parameters reflect their preparedness for surviving their first winter's fast. During the surveys there were distinct regional and interannual variations in all three parameters for individual groups of herring in Prince William Sound. Likewise, with each collection there was typically a large range of size and WBEC values. The average standard length was (±S.D.) 80±13 mm (range=40–118), the mean whole body wet weight was 5·7±3·0 g (range=0·7–29·2) and the average WBEC of all age-0 herring captured, regardless of year or site (n=1471), was 5·4± 1·0 kJ g⁻¹ wet weight (range=2·4–9·4). The large range of WBEC and size indicates that age-0 herring at different capture sites were not all equally prepared for surviving their first winter.     

Trophic ecology of Squatina guggenheim on the continental shelf off Uruguay and northern Argentina

A total of 1280 (670 females and 610 males) Squatina guggenheim , an angel shark endemic to the south-west Atlantic, was caught during five research cruises (1995, 1997 and 1998) carried out in the Argentine-Uruguayan Common Fishing Zone (AUCFZ) at depths of 3·5–266 m. All angel sharks captured were sexed and measured. Of the 947 examined for stomach contents, 556 (58·7%) contained food and 391 (41·3%) were empty. Numerically, bony fishes were the principal dietary component (89·7%), followed by crustaceans (4·8%) and molluscs (4·4%). The most important prey species were Engraulis anchoita (58·3%), shrimps (4·4%) and Ilex argentinus (2·3%). Male and female diets were not found to differ significantly ( P  > 0·01), however, the diets of different size classes showed significant differences ( P  ≤ 0·01) (group 1, 23–44, group 2, 45–74 and group 3, 75–91 cm L _T). Low rates of cannibalism were observed. The spatial and seasonal variability in the trophodynamics of S. guggenheim is discussed.     

Long-term cryopreservation of sperm of Japanese eel

After cryopreservation in a diluent (D#5-D), containing 76·2 mM NaHCO3, 137·0 mM NaCl, 1·4% soya lecithin and 10% dimethyl sulphoxide, for 30–180 days, Japanese eel Anguilla japonica sperm was 61·4–70·6% motile compared with fresh sperm. Cryopreserved sperm was used for fertilizing three batches of eggs and hatchability ranged from 20·7 to 69·1%. These results indicate that D#5-D is an useful diluent for sperm cryopreservation in Japanese eel.     

Some morphometric and biochemical features of ready-to-migrate silver and pre-migratory yellow stages of the shortfin eel of south-eastern Australian waters

The mean total length ( L _T), mass and age of ready to migrate female silver shortfin eels Anguilla australis from the Hopkins River estuary and the mouth of the Merri River in south-eastern Australia, were 83·2 ± 1·2 cm, 1051 ± 51 g, and 17·2 ± 1·79 years, respectively. The eye index ( I _E) of the silver shortfin eels was < 5·2 (mean 7·64 ± 0·29) and differed significantly from that of the yellow shortfin eels collected from two other sites. The I _E increased with L _T (mm) and was related by log I _E= 2·656 log L _T6·925. The per cent moisture, protein and ash content of the liver of silver shortfin eels was significantly lower than in yellow shortfin eels, but lipid content was significantly higher in the former (35·5 ± 2·0%). The mean mass μg mg lipid ⁻) of saturates (230·4 ± 2·6 v . 181·7 ±2·6), monoenes (367·4 ± 6·3 v . 290·8 ± 8·9) and PUFA (177·3 ± 5·3 v . 159·7 ± 4·6) in muscle was significantly higher, and the great majority of individual fatty acids was found also in higher quantities in silver shortfin eels. In the liver, the PUFA found in the highest quantity was 22:6n-3, except in shortfin eels from Hopkins River estuary, and the amount of 18:2n-6 in the liver of silver shortfin eels was significantly higher than that in yellow shortfin eels but the reverse was true of 20:4n-6. In both muscle and liver tissues the saturate 16:0 and the monoene 18:ln-9 collectively accounted for >50% of all the fatty acids in the lipid.     

Occurrence of D-amino acids in a few archaea and dehydrogenase activities in hyperthermophile Pyrobaculum islandicum

The contents of D-enantiomers of serine, alanine, proline, glutamate (glutamine) and aspartate (asparagine) were examined in the membrane fractions, soluble proteins and free amino acids from some species of archaea, Pyrobaculum islandicum, Methanosarcina barkeri and Halobacterium salinarium. Around 2% (D/D+L) of D-aspartate was found in the membrane fractions. In the soluble proteins, the D-amino acid content was higher in P. islandicum than that in the other archaeal cells: the concentrations in P. islandicum were 3 and 4% for D-serine and D-aspartate, respectively. High concentrations of free D-amino acids were found in P. islandicum and H. salinarium; the concentrations of D-serine (12-13%), D-aspartate (4-7%) and D-proline (3-4%) were higher than those of D-alanine and D-glutamate. This result showed a resemblance between these archaea and not bacterial, but eukaryotic cells. The presence of D-amino acids was confirmed by their digestion with D-amino acid oxidase and D-aspartate oxidase. The occurrence of D-amino acids was also confirmed by the presence of activities catalyzing catabolism of D-amino acids in the P. islandicum homogenate, as measured by 2-oxo acid formation. The catalytic activities oxidizing D-alanine, D-aspartate and D-serine at 90 degrees C were considerably high. Under anaerobic conditions, dehydrogenase activities of the homogenate were 69, 84 and 30% of the above oxidase activities toward D-alanine, D-aspartate and D-serine, respectively. Comparable or higher dehydrogenase activities were also detected with these D-amino acids as substrate by the reduction of 2, 6-dichlorophenolindophenol. No D-amino acid oxidase activity was detected in the homogenates of M. barkeri and H. salinarium.     

Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: efficient regeneration of the fusion protein and selective cleavage with trypsin.

Bacteriorhodopsin (bR) is a light-driven proton pump from Halobacterium salinarium and is a model system for studying membrane protein folding, stability, function, and structure. bR is composed of bacterio-opsin (bO), the 248-amino acid apo protein, and all-trans retinal, which is linked to lysine 216 via a protonated Schiff base. A bO gene (sbOd) possessing 29 unique restriction sites and a carboxyl-terminal purification epitope (1D4, nine amino acids) has been designed and synthesized. Overexpression of bO was achieved by fusion to the carboxyl terminus of maltose binding protein (MBP). The expressed fusion protein (MBP-sbO-1D4) formed inclusion bodies in Escherichia coli and, following solubilization with urea and removal of the urea by dialysis, approximately 170 mg of approximately 75% pure MBP-sbO-1D4 was obtained from 1 L of culture. MBP-sbO-1D4 formed high molecular weight (> or = 2,000 kDa) oligomers that were water-soluble. The synthetic bO with the 1D4 tag (sbO-1D4) was separated from MBP by trypsin cleavage at the factor Xa site between the MBP and sbO-1D4 domains. Selective trypsin cleavage at the factor Xa site, instead of at the 14 other potential trypsin sites within bO, was accomplished by optimization of the digestion conditions. Both MBP-sbO-1D4 and sbO-1D4 were regenerated with all-trans retinal and purified to homogeneity. In general, 6-10 mg of sbR-1D4 and 52 mg of MBP-sbR-1D4 were obtained from 1 L of cell culture. No significant differences in terms of UV/vis light absorbance, light/dark adaptation, and photocycle properties were observed among sbR-1D4, MBP-sbR-1D4, and bR from H. salinarium.     

Cytokinesis of Candida albicans by Diethylstilbestrol

In vitro effects of the synthetic oestrogenic hormone diethylstilbestrol (DS) and diethylstilbestrol dipropionate (DSP) on Candida albicans have been assessed. At a concentration of 5–20 μg/ml. these compounds suppressed the growth of C. albicans even though the multiplication of the organism was not influenced immediately. When C. albicans was grown for approximately 4 h in tryptic soy broth, its multiplication was rapidly retarded by these two substances. Since C. albicans must grow on a suitable culture medium in order to absorb DS and DSP, it was not surprising that respiration, the uptake, and incorporation of nutrients by C. albicans was not influenced when the cells were 'resting'. Such plasma steroids as androsterone (0·5 μg/ml), 5α-androstan-3 β-diol (0·25 μg/ml), dehydroisoandrosterone (2 μg/ml), epiandrosterone (0·1 μg/ml), oestrone (0·1 μg/ml), progesterone (0·4 μg/ml), cortisol (0·2 μg/ml), cholesterol (10 μg/ml) in combination with DSP did not antagonize the retardive action of DSP for C. albicans .     

Reproductive investment in the Silurus meridionalis

A comparison of pre- and postspawning Silurus meridionalis showed that 20·7% of body stored energy was utilized during spawning for a standard male (74·5 cm) and 23·8% for a standard female (85·3 cm). About one-third of the loss of the stored energy was released as eggs by females, and almost all of the energy loss for males and about two-thirds for females were expended in metabolism. Stored lipid as fuel for metabolism supplied 90·0% of energy in males and 95·2% in females, and protein supplied the rest of the energy. Models for predicting energy in released gametes ( G _g), deposited in the body as somatic growth ( G _s), utilized in spawning activity ( S _a), expended in maintenance ( M , including metabolism, faeces and excretion), and food energy ( C ) were developed, and annual energy budgets were compiled. The balanced budget for a male aged 4 was: 100 C =0·06 G _g+11·17 S _a+19·5 G _s+69·2 M , and for a female aged 5: 100 C =5·48 G _g+8·51 S _a+15·8 G _s+70·2 M .