Production of functional agarolytic nano-complex for the synergistic hydrolysis of marine biomass and its potential application in carbohydrate-binding module-utilizing one-step purification

Marine algal polysaccharides are promising alternative resources to terrestrial biomass. Agarolytic enzymes degrade agarose to various kinds of oligosaccharides. In this study, we expressed miniCbpA, a recombinant scaffolding protein from Clostridium cellulovorans, in Escherichia coli. To assemble the agarolytic complex via cohesin–dockerin interaction, we constructed a chimeric agarase cAgaB from Zobellia galactanivorans containing the catalytic domain of AgaB fused with a dockerin domain from C. cellulovorans EngB. The assembly of functional agarolytic complexes increased the activity against the agar substrate approximately 1.4-fold compared with that for the corresponding enzymes alone. The carbohydrate-binding module (CBM) of miniCbpA was used as a tag for CBM-utilizing one-step purification using cellulose as a support. This is the first report on the formation of agarolytic complexes using the cohesin–dockerin interaction system. The assembly of agar-degrading complexes will lead to the commercial production of useful products from agar biomass at low costs.     

Genomic and Proteomic Analyses of the Agarolytic System Expressed by Saccharophagus degradans 2-40

Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S. degradans 2-40 agarolytic system were identified. Five agarases were identified in the S. degradans 2-40 genome. Aga50A and Aga50D include GH50 domains. Aga86C and Aga86E contain GH86 domains, whereas Aga16B carries a GH16 domain. Novel family 6 carbohydrate binding modules (CBM6) were identified in Aga16B and Aga86E. Aga86C has an amino-terminal acylation site, suggesting that it is surface associated. Aga16B, Aga86C, and Aga86E were detected by mass spectrometry in agarolytic fractions obtained from culture filtrates of agar-grown cells. Deletion analysis revealed that aga50A and aga86E were essential for the metabolism of agarose. Aga16B was shown to endolytically degrade agarose to release neoagarotetraose, similarly to a β-agarase I, whereas Aga86E was demonstrated to exolytically degrade agarose to form neoagarobiose. The agarolytic system of S. degradans 2-40 is thus predicted to be composed of a secreted endo-acting GH16-dependent depolymerase, a surface-associated GH50-dependent depolymerase, an exo-acting GH86-dependent agarase, and an α-neoagarobiose hydrolase to release galactose from agarose.     

Pasteurella Pseudotuberculosis Infection in Man

Pasteurella pseudotuberculosis has been considered a widespread animal pathogen for many years, but only within the last decade has its capacity to cause human disease been recognized. Two forms of human disease have been established—acute septicemia and mesenteric lymphadenitis. Because mesenteric adenitis is frequently indistinguishable from acute appendicitis, blood serum was obtained from 66 consecutive patients who underwent operation for appendicitis and was examined for agglutinins to seven serotype strains of P. pseudotuberculosis. Agglutinins were obtained in 21.2% of this series. Titres of over 1/100 were found in three of three cases of mesenteric lymphadenitis, one of 11 with no apparent disease, and one of 46 with appendicitis. P. pseudotuberculosis was isolated from a lymph node in the latter case. Two to four follow-up samples of sera in each of these five cases had increasing and then decreasing titres, indicative of active disease. Titres of 1/15 or less were found in five of the cases of appendicitis, in one case of salpingitis, and in three with no apparent disease. The occurrence of these nine cases with low titres may be indicative of previous contact with the organism.Human infection with P. pseudotuberculosis is not unusual in the Edmonton region and is responsible for at least some cases of mesenteric lymphadenitis.     

The IJsselmeer and its phytoplankton--with special attention to the suitability of the lake as a habitat for Oscillatoria agardhii Gom

The IJsselmeer (surface area 1200 km², mean depth 4.5 m, residencetime 0.4 year, phosphorus load 7 g m^–2 year^–1) isa very important conservation area. Regular summer bloomingof Oscillatoria spp. can depreciate this value, so the boundsof possibility of this kind of blooming have been investigated.Therefore samples were taken along the shore and in the openwater from 1974 to 1982, continuous temperature profile measurementswere made in the same period in the middle of the lake and insitu primary production was measured in 1976 and 1977. The phytoplanktonconsists of green algae throughout the year, diatoms in spring,and blooms of Microcystis aeruginosa in summer. Blooming ofOscillatoria agardhii Gom occurs regularly in summer along theFrisian shore. In 1976, however, a heavy bloom of this algaoccurred in the whole lake. Comparing the IJsselmeer with shallowerOscillatoria-lakes in the Netherlands distinct differences arepresent concerning biomass, chlorophyll a content, relativevolume of the euphotic zone and light-dark cycle. Not only thelarger depth and extensiveness of the IJsselmeer are unfavourablefactors for Oscillatoria, but also the separation by land reclamationof many of the shallow littoral regions from the main body ofthe lake. High temperature and microstratification are neededto develop a bloom in the whole lake.     

Taxon composition and genetic variation of water frogs in the Mid-Rhone floodplain

Natural hemidonal hybrid lineages of water frogs reproduce by hybridogenesis, excluding one parental genome in the germ line and mating with the coexisting same parental species. Two such sexual hosthybridogen systems occur in the Rhône valley: the L-E system in the north, the P-G system in the south. Although these hybridogenetic complexes may overlap along the Rhône river, there is no evidence for a contact zone in our samples: only Rana ridibunda and R. esculenta were identified using protein electrophoresis. Whether the absence of R. perezi reflects a more southern distribution or its exclusive occurrence in other habitats, remains to be tested. Comparison of somatic and gonadal tissues reveals that gametogenesis of R. esculenta is of the L-E type: gametes carry ridibunda genomes. R. ridibunda apparently is not native, but was introduced by humans, and the R. esculenta in our samples is probably an immigrant from nearby L-E systems.     

Incidence of Listeria monocytogenes in Nature

During a research project on the occurrence of Listeria monocytogenes 194 strains were isolated in southern West Germany during the years 1972 to 1974: 154 from soil and plant samples (20.3%), 16 from feces of deer and stag (15.7%), 9 from old moldy fodder and wildlife feeding grounds (27.2%), and 8 from birds (17.3%). The highest number of isolates was obtained from uncultivated fields. The beta-hemolytic serovars 1/2b and 4b were predominant; other serovars (some of them identified for the first time), including nonhemolyzing strains, have been encountered frequently. It is suggested that Listeria monocytogenes is a saprophytic organism which lives in a plant-soil environment and therefore can be contracted by humans and animals via many possible routes from many sources.     

The relation of biotic and abiotic interactions to eutrophication in Tjeukemeer,The Netherlands

During the summer months of 1974–1985 chlorophyll-a and total P concentration, biomass of Daphnia hyalina, smelt Osmerus eperlanus, bream Abramis brema and pikeperch Stizostedion lucioperca, water temperature and water intake from lake IJsselmeer were monitored in Tjeukemeer. During this period there were manipulations with the bream and pikeperch stocks as a consequence of the termination of a gill-net fishery in 1977, and larval smelt immigrated each year from the large lake IJsselmeer and contributed largely to the yearly smelt recruitment.The correlation matrix of the nine variables mentioned above showed a positive correlation between bream and chlorophyll-a, but surprisingly a negative one between smelt and chlorophyll-a. The latter can only be explained when smelt is the dependent variable. In a multi-linear regression there was a negative effect of temperature, chlorophyll a and pikeperch on smelt and a positive effect of water intake. Daphnia hyalina was negatively influenced by the biomass of smelt and the water intake of lake IJsselmeer. The positive relation of Daphnia hyalina and chlorophyll-a was probably related to better survival chances of D. hyalina in an Oscillatoria-rich environment when smelt is the most important predator. An increasing biomass of bream coincided with higher total-P levels and probably contributed to higher chlorophyll-a levels.     

Phosphorus eutrophication in the SW Frisian lake district 2. Phosphorus balances and simulation of reduction scenarios

The lakes and interconnecting canals in the S.W. Frisian lake district are highly eutrophic. In the mid-1980's a project on eutrophication and lake restoration research was started. This project was aimed at modelling water transport and phosphorus (P) dynamics and at simulating management scenarios. A simple dynamic P-balance model was used to calculate total phosphorus (TP) balances and to simulate three TP reduction scenarios in each of three lakes: Tjeukemeer, Groote Brekken and Slotermeer. The model covered three periods in 1985, 1986 and 1987. The external loads to Tjeukemeer were highest, moderate to Groote Brekken, and lowest to Slotermeer. The major P sources in the area were discharges from the surrounding polders, used mainly for agriculture, and from IJsselmeer.Despite a 75% TP-reduction in water from the surrounding polders the 0.07 mg l^–1 target level could be reached only occasionally in Tjeukemeer, while in the other two lakes this level was not even approached. The effect of a 75% reduction in water from IJsselmeer was greatest in Groote Brekken (but again approached the target only occasionally), moderate in Tjeukemeer and least in Slotermeer. The simulations showed that only a 75% reduction in both external loads (from polders and from IJsselmeer) will lead to achieving the target level in Tjeukemeer and Groote Brekken during the summer periods. In Slotermeer, a relatively isolated lake, other measures are necessary to reach the target level. The results are confirmed by an approximate theoretical analysis of the effects of load reduction.     

Distribution ofVibrio cholerae in two Florida estuaries

The distribution ofVibrio cholerae was examined in 2 Florida estuaries, Apalachicola and Tampa Bay.Vibrio cholerae serotype non-01 was the most abundant serotype, being isolated from 45% of the oyster samples, 30% of the sediments, 50% of the waters, and 75% of the blue crabs.Vibrio cholerae serotype 01 was isolated from only one oyster sample. Strong linear correlations betweenV. cholerae and temperature, salinity, or the other physical/chemical parameters measured,Escherichia coli, or fecal coliforms were not observed, but a range of temperatures and salinities appeared relevant to the distribution of the organism. The organism was present in the highest concentrations when salinities were 10‰–25‰ and temperatures were 20?C–35?C.In vitro growth curves of 95V. cholerae environmental isolates further supported that 10‰–25‰ was an ideal salinity range for the organisms. The results suggest thatV. cholerae is a widely distributed organism in the nutrient-rich warm waters of the Gulf Coast estuaries.     

Blastocystis subtype 5: Predominant subtype on pig farms,Thailand

Blastocystis is a unicellular protist most commonly detected in humans and a variety of animals. The predominant mode of its transmission is the fecal–oral route, but its zoonotic potential is not completely understood. The objective of this study was to determine the presence and genetic diversity of Blastocystis on pig farms in Nakhon Pathom Province, Central Thailand. A total of 154 human and 90 pig stool samples were collected and analyzed. Nested PCR detected Blastocystis in 35.55% of the pig samples and 6.49% of the human samples. Subtyping based on regions of the small-subunit ribosomal RNA (SSU rRNA) gene identified three Blastocystis subtypes in pigs and humans: ST1, ST3, and ST5. Blastocystis ST5 was the predominant subtype, followed by ST1 and then ST3. All the sequences from the Blastocystis-positive samples from both pigs and humans were closely related. This study reveals a possibility of low host specificity of Blastocystis STs (ST1, ST3 and ST5) on pig farms in Thailand. We tentatively suggest that close contact with or exposure to pig stools may be a significant source of Blastocystis detected in pig handlers. Further studies are required to confirm the zoonotic transmission of this organism in Thailand, because pigs may play an important role in the transmission of Blastocystis.     

Marine elements in fish fauna of southeastern Ethiopia

The eel Anguilla bengalensis labiata (Peters) is reported from the middle reaches of the Genale River drainage in southeastern Ethiopia based on our own samples, as well as, on prevously undesribed or poorly described samples obtained by earlier investigators. The Anguilla eels from the Indian Ocean appeared to be the only group of diadromous fishes in Ethiopian fauna. An occurrence of the goby Glossogobius giuris (Hamilton) in the same drainage is reported based on four specimens from the lower reaches of the Genale River. Gross morphology of Ethiopian eels and gobies was described in relation to their identification. The distribution of other saltwater dispersants in the Ethiopian waters is briefly discussed.     

Diatom-inferred trophic history of IJsselmeer (The Netherlands)

IJsselmeer was formed in 1932 through the closure of the Afsluitdijk that separated the artificial lake from the former Zuiderzee estuary. The palaeoecology of IJsselmeer was studied on a 63-cm-long sediment core. Lithology and microfossil data, particularly the diatom flora, clearly show the transition from the marine Zuiderzee into the freshwater IJsselmeer. Trophic conditions in IJsselmeer since 1932 have been inferred by qualitative and quantitative diatom-based approaches: by plotting the distribution of trophic categories based on published trophic indicator values, by a canonical correspondence analysis (CCA) yielding relative total phosphorus (TP) changes and by applying a transfer function in order to calculate TP concentrations. All three approaches indicate that IJsselmeer initially was meso- to eutrophic. A first hypertrophic period is indicated for the mid-1940s, likely due to internal loading. After 1960, the phosphorus load steadily increased and TP in IJsselmeer reached highest concentrations (ca. 150 μg l⁻¹) in the 1980s as confirmed by monitoring data since 1975. The monitored data show that the TP concentration continuously decreased after 1985 due to successful environmental protection measures. This trend is not (or not yet) evidenced by the diatom data and thus, the diatom-inferred TP concentration. Guest editors: K. Buczkó, J. Korponai, J. Padisák & S. W. Starratt Palaeolimnological Proxies as Tools of Environmental Reconstruction in Fresh Water     

Transformation of 1-Menthene by a Cladosporium: Accumulation of beta-Isopropyl Glutaric Acid in the Growth Medium

An organism isolated from soil samples collected near a terpene plant grew in the presence of 1-menthene, with no other major source of carbon and energy. The organism was tentatively identified as a member of the genus Cladosporium. When this organism was grown in the presence of 1-menthene, with no other major source of carbon, substantial quantities of β-isopropyl glutaric acid accumulated in the fermentation medium. β-Isopropyl glutaric acid is probably an end product of the degradation of 1-menthene by the organism.     

Transformation of 1-Menthene by a Cladosporium: Accumulation of β-Isopropyl Glutaric Acid in the Growth Medium

An organism isolated from soil samples collected near a terpene plant grew in the presence of 1-menthene, with no other major source of carbon and energy. The organism was tentatively identified as a member of the genus Cladosporium. When this organism was grown in the presence of 1-menthene, with no other major source of carbon, substantial quantities of β-isopropyl glutaric acid accumulated in the fermentation medium. β-Isopropyl glutaric acid is probably an end product of the degradation of 1-menthene by the organism.     

Occurrence of Nontuberculous Mycobacteria in Environmental Samples

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infection in immunocompromised hosts. Because there is no evidence of person-to-person transmission and NTM have been found in drinking water, the environment is considered a likely source of infection. In this study the widespread occurrence of NTM was examined in drinking water, bottled water, and ice samples. A total of 139 samples were examined for NTM by a membrane filtration culture technique followed by PCR amplification and 16S rRNA sequence determination to identify the isolates. NTM were not detected in bottled water or cisterns but were detected in 54% of the ice samples and 35% of the public drinking-water samples from 21 states. The most frequently occurring isolate was M. mucogenicum (formerly referred to as an M. chelonae-like organism).     

Seasonal Occurrence and Distribution of Microbial Indicators and Pathogens in the Rhode River of Chesapeake Bay

The seasonal incidence and occurrence of indicator organisms and pathogens were studied at four sites in the Rhode River, a subestuary of Chesapeake Bay. The highest frequency of occurrence of total and fecal coliforms and fecal streptococci was in Muddy Creek, a marsh area receiving pasture land runoff. Second highest frequency of occurrence of these bacteria was in Cadle Creek, a populated area. Lowest measurements of these parameters were obtained at stations in the central portion of the Rhode River. No Salmonella spp. were detected by the methods employed in this study. However, it is concluded that if these organisms are present, the concentrations are ≤1 organism per liter. The presence of Clostridium botulinum was detected in 12% of the samples tested.     

Connectivity between Migrating and Landlocked Populations of a Diadromous Fish Species Investigated Using Otolith Microchemistry

Smelt Osmerus eperlanus has two different life history strategies in the Netherlands. The migrating population inhabits the Wadden Sea and spawns in freshwater areas. After the closure of the Afsluitdijk in 1932, part of the smelt population became landlocked. The fresh water smelt population has been in severe decline since 1990, and has strongly negatively impacted the numbers of piscivorous water birds relying on smelt as their main prey. The lakes that were formed after the dike closure, IJsselmeer and Markermeer have been assigned as Natura 2000 sites, based on their importance for (among others) piscivorous water birds. Because of the declining fresh water smelt population, the question arose whether this population is still supported by the diadromous population. Opportunities for exchange between fresh water and the sea are however limited to discharge sluices. The relationship between the diadromous and landlocked smelt population was analysed by means of otolith microchemistry. Our interpretation of otolith strontium (⁸⁸Sr) patterns from smelt specimens collected in the fresh water area of Lake IJsselmeer and Markermeer, compared to those collected in the nearby marine environment, is that there is currently no evidence for a substantial contribution from the diadromous population to the spawning stock of the landlocked population.     

Agar-degrading bacteria isolated from Antarctic macroalgae

This study describes the taxonomic diversity of pigmented, agar-degrading bacteria isolated from the surface of macroalgae collected in King George Island, Antarctica. A total of 30 pigmented, agarolytic bacteria were isolated from the surface of the Antarctic macroalgae Adenocystis utricularis, Monostroma hariotii, Iridaea cordata, and Pantoneura plocamioides. Based on the 16S rRNA data, the agarolytic isolates were affiliated to the genera Algibacter, Arthrobacter, Brachybacterium, Cellulophaga, Citricoccus, Labedella, Microbacterium, Micrococcus, Salinibacterium, Sanguibacter, and Zobellia. Isolates phylogenetically related to Cellulophaga algicola showed the highest agarase activity in culture supernatants when tested at 4 and 37 °C. This is the first investigation of pigmented agar-degrading bacteria, members of microbial communities associated with Antarctic macroalgae, and the results suggest that they represent a potential source of cold-adapted agarases of possible biotechnological interest.     

Medium for Isolation and Differentiation of Vibrio parahaemolyticus and Vibrio alginolyticus

Trypticase soy agar supplemented with sucrose, sodium chloride, bile salts, and triphenyltetrazolium chloride is an improved plating medium for the isolation of Vibrio parahaemolyticus from samples of seawater, permitting better differentiation of this organism from Vibrio alginolyticus and other bacteria.