Comparative study of the aerobic, heterotrophic bacterial flora of Chesapeake Bay and Tokyo Bay.

A comparative study of the bacterial flora of the water of Chesapeake Bay and Tokyo Bay was undertaken to assess similarities and differences between the autochthonous flora of the two geographical sites and to test the hypothesis that, given similarities in environmental parameters, similar bacterial populations will be found, despite extreme geographic distance between locations. A total of 195 aerobic, heterotrophic bacterial strains isolated from Chesapeake Bay and Tokyo Bay water were examined for 115 biochemical, cultural, morphological, nutritional, and physiological characters. The data were analyzed by the methods of numerical taxonomy. From sorted similarity matrices, 77% of the isolates could be grouped into 30 phena and presumptively identified as Acinetobacter-Moraxella, Caulobacter, coryneforms, Pseudomonas, and Vibrio spp. Vibrio and Acinetobacter species were found to be common in the estuarine waters of Chesapeake Bay, whereas Acinetobacter-Moraxella and Caulobacter predominated in Tokyo Bay waters, at the sites sampled in the study.     

Comparative study of the aerobic, heterotrophic bacterial flora of Chesapeake Bay and Tokyo Bay.

A comparative study of the bacterial flora of the water of Chesapeake Bay and Tokyo Bay was undertaken to assess similarities and differences between the autochthonous flora of the two geographical sites and to test the hypothesis that, given similarities in environmental parameters, similar bacterial populations will be found, despite extreme geographic distance between locations. A total of 195 aerobic, heterotrophic bacterial strains isolated from Chesapeake Bay and Tokyo Bay water were examined for 115 biochemical, cultural, morphological, nutritional, and physiological characters. The data were analyzed by the methods of numerical taxonomy. From sorted similarity matrices, 77% of the isolates could be grouped into 30 phena and presumptively identified as Acinetobacter-Moraxella, Caulobacter, coryneforms, Pseudomonas, and Vibrio spp. Vibrio and Acinetobacter species were found to be common in the estuarine waters of Chesapeake Bay, whereas Acinetobacter-Moraxella and Caulobacter predominated in Tokyo Bay waters, at the sites sampled in the study.     

Occurrence ofVibrio and other bacteria on the sea nettle,Chrysaora quinquecirrha

Sixty-two specimens of the sea nettle,Chrysaora quinquecirrha, were caught in the lower Chesapeake Bay, homogenized, and samples plated on a yeast extract-Bay water agar. Bacterial colonies were selected randomly, purified, and tested for 180 characteristics. Computer analysis permitted clustering of the 208 isolates into 15 groups (comprised of 133 strains) plus 75 nongrouped strains which failed to associate with any group at the 70% similarity level. The majority of the isolates (68.8%) wereVibrio species. These included 110 of the grouped strains (forming 12 of the 15 groups) and 33 of the nongrouped strains. The remainder of the isolates were distributed as follows:Pseudomonas (11.6%),Bacillus (8.2%),Flavobacterium (2.4%),Acinetobacter (2.4%),Moraxella (1.9%),Cytophaga (1.9%), Gram-positive cocci (1.4%), and miscellaneous (1.4%). All theBacillus were isolated from a group of moribund nettles and reflect an abnormal condition.Vibrio species predominated in the five catches of healthy nettles, but were distinctly different for each catch.     

Characteristics of bacterium GB-2, a presumptiveCytophaga species with novel immunoregulatory properties

A Gram-negative, psychrophilic bacterium, designated GB-2, was isolated from fetal calf serum and analyzed for its morphological, physiological, and biochemical characteristics. Gliding motility, sensitivity to actinomycin D, the presence of the pigment flexirubin and cytochrome c, growth on a variety of carbohydrates, the production of acid fermentation products, and a 34.9 mol% guanine+cytosine (G+C) content of bacterial DNA indicate GB-2 to be a member either of the generaCytophaga orFlexibacter. Growth of GB-2 was optimized in a simple defined medium to facilitate isolation and characterization of bacterial products. Liquid growth of GB-2 resulted in the release of significant quantities of a macromolecule free of both endotoxin and protein into the growth supernatant, which activated the proliferation of murine lymphocytes. The relationship between this bacterium and its end-products to other species of theCytophaga/Flexibacter group is discussed.     

Numerical taxonomy of heavy metal-tolerant bacteria isolated from an estuary.

A total of 230 strains of metal-tolerant bacteria from water and sediment samples collected in Chesapeake Bay were isolated on a medium containing cobalt, lead, mercury, or molybdenum. In addition, a set of 71 cultures were simultaneously isolated on glucose tryptone yeast extract agar medium without metals. Twenty-three reference strains were also included in the numerical taxonomy study of these bacteria, bringing the grand total of strains examined to 324. All strains were examined for 112 biochemical, cultural, morphological, and physiological characters. The taxonomic data obtained were analyzed by computer and the simple matching (SSM) and Jaccard (SJ) coefficients were calculated. Clustering achieved by unweighted average linkage is presented and, from sorted similarity matrices and dendrograms, 294 strains, i.e., 97% of the total, were recovered in 12 phenetic groups defined at the 75 to 80% similarity level. Among the strains there were nine phena presumptively identified as Bacillus, Erwinia, Mycobacterium, Pseudomonas, and coryneforms. From the results of the taxonomic study, it is concluded that metal tolerance in estuarine water and sediment bacteria occurs among a restricted range of taxa distributed throughout the estuarine environment.     

Cytophaga xylanolytica sp. nov., a xylan-degrading,anaerobic gliding bacterium

Gliding bacteria attached in masses to, and dominated the fermentation of, xylan powder in methanogenic and sulfidogenic enrichments from various freshwater sediments. Isolates of such bacteria were all gram-negative, slender rods (0.4×4-24 m) that formed no endospores, microcysts or fruiting bodies. Representative strain XM3 was a mesophilic, aeroduric anaerobe that grew by fermentation of mono-, di-, and poly-saccharides (but not cellulose) in a mineral medium containing up to 3% NaCl. However, CO₂/HCO _inf3 ^sup- was required in media for consistent initiation of growth. Fermentation products included acetate, propionate, succinate, CO₂, and H₂. Xylan-grown cells had xylanase and various glycosidase activities that were mainly or almost entirely cell-associated, respectively. Strain XM3 was weakly catalase positive, but oxidase negative; it possessed sulphonolipids and carotenoid, but not flexirubin, pigments; and its total cellular fatty acids were dominated by C_15:0 anteiso (75%), n (13%) and iso (2%) isomers. Strain XM3 had 45.5 mol% G+C in its DNA, and partial sequencing of its 16S rRNA placed XM3 within the Bacteroides-Flavobacterium phylogenetic group. Similar strains were isolated from marine sediments. Strain XM3 is herewith proposed as the type strain of the new species, Cytophaga xylanolytica. Results, which are discussed in terms of our current concept of the genus Cytophaga, suggest that the importance of C. xylanolytica in anaerobic biopolymer decomposition has not been fully appreciated.Dedicated to Professor Norbert Pfennig, in whose laboratory this project was initiated and who recently retired after more than four decades of contributions to microbiology     

Grazing by protozoa as selection factor for activated sludge bacteria

In continuous culture enrichments that were inoculated with activated sludge and were fed with polymeric substrates, freely dispersed single-celled bacteria belonging to theCytophaga group dominated among the initial populations, irrespective of the activated sludge source. These populations were grazed by flagellated protozoa which after several days reached high cell densities. Other morphologic bacterial groups such as spiral-shaped or filamentous bacteria then became dominant. In defined mixed culture experiments with bacterial isolates from the enrichment cultures, it was shown that a grazing-resistantMicrocyclus strain outgrew aCytophaga strain in the presence of grazing protozoa. In contrast, theCytophaga strain competed successfully with theMicrocyclus strain and with other grazing-resistant strains under protozoa-free conditions. Furthermore, it was demonstrated that assumed grazing resistance factors such as floccing or filamentous growth were lost by some of the strains when they were grown for several generations in continuous culture under the same conditions, but in the absence of protozoa.     

Numerical taxonomy of phenanthrene-degrading bacteria isolated from the Chesapeake Bay.

Phenanthrene-degrading bacteria were isolated from Chesapeake Bay samples by the use of a solid medium which had been overlaid with an ethanol solution of phenanthrene before inoculation. Eighteen representative strains of phenanthrene-degrading bacteria with 21 type and reference bacteria were examined for 123 characteristics representing physiological, biochemical, and nutritional properties. Relationships between strains were computed with several similarity coefficients. The phenogram constructed by unweighted-pair-group arithmetic average linkage and use of the simple Jaccard (SJ) coefficient was used to identify seven phena. Phenanthrene-degrading bacteria were identified as Vibrio parahaemolyticus and Vibrio fluvialis by their clustering with type and reference strains. Several phenanthrene-degrading bacteria resembled Enterobacteriaceae family members, although some Vibrio-like phenanthrene degraders could not be identified.     

Numerical taxonomy of phenanthrene-degrading bacteria isolated from the Chesapeake Bay

Phenanthrene-degrading bacteria were isolated from Chesapeake Bay samples by the use of a solid medium which had been overlaid with an ethanol solution of phenanthrene before inoculation. Eighteen representative strains of phenanthrene-degrading bacteria with 21 type and reference bacteria were examined for 123 characteristics representing physiological, biochemical, and nutritional properties. Relationships between strains were computed with several similarity coefficients. The phenogram constructed by unweighted-pair-group arithmetic average linkage and use of the simple Jaccard (SJ) coefficient was used to identify seven phena. Phenanthrene-degrading bacteria were identified as Vibrio parahaemolyticus and Vibrio fluvialis by their clustering with type and reference strains. Several phenanthrene-degrading bacteria resembled Enterobacteriaceae family members, although some Vibrio-like phenanthrene degraders could not be identified.     

Occurrence of Cytophagas in Sewage Plants

With the application of plate count methods and of the KOH-flexirubin test, bacteria belonging to the Cytophaga group were proved to occur regularly in samples from biological sewage treatment facilities. Generally, the percentage of Cytophaga colonies of the total heterotrophic colonies was lowest in the inflow sewage water as compared with the values found in activated sludge, trickling filter, and effluent samples. During an observation period of 16 months, the highest percentages of cytophagas were found in winter samples from activated sludge and trickling filters. Furthermore, cytophagas were shown to have high percentages of the bacteria lytic to polymeric substrates such as cellulose, chitin, dextran, pectin, xylan, and gelatin. Thus, it is suggested that cytophagas may contribute to sewage purification, especially at cold temperatures and by polymer breakdown. Cytophaga strains isolated were shown to have gliding motility, flexirubin pigmentation, and a low guanine plus cytosine base ratio in common. The strains were roughly subdivided into a spreading, a nonspreading, and a cellulolytic group.     

Comparative Study of Microbial Communities from Cultured and Natural Populations of the Mussel Mytilus trossulus in Peter the Great Bay

The 525 strains of heterotrophic bacteria isolated from natural and cultured populations of the mussel Mytilus trossulus and the surrounding seawater were identified to a genus level on the basis of phenotypic analysis and the fatty acid composition of cell lipids. Gram-negative isolates were dominated by six genera of the family Enterobacteriaceae and by the genera Pseudoalteromonas, Vibrio, Photobacterium, Cytophaga/Flavobacterium/Bacteroides, Pseudomonas, and Moraxella. Gram-positive isolates were mainly represented by the genus Streptomyces. The taxonomic compositions of natural and cultured populations of the mussel M. trossulus in Peter the Great Bay were similar.     

Ecology, serology, and enterotoxin production of Vibrio cholerae in Chesapeake Bay.

A total of 65 isolates of Vibrio cholerae, serotypes other than O--1, have been recovered from water, sediment, and shellfish samples from the Chesapeake Bay. Isolations were not random, but followed a distinct pattern in which salinity appeared to be a controlling factor in V. cholerae distribution. Water salinity at stations yielding V. cholerae (13 out of 21 stations) was 4 to 17 0/00, whereas the salinity of water at stations from which V. cholerae organisms were not isolated was less than 4 or greater than 17 0/00. From results of statistical analyses, no correlation between incidence of fecal coliforms and V. cholerae could be detected, whereas incidence of Salmonella species, measured concurrently, was clearly correlated with fecal coliforms, with Salmonella isolated only in areas of high fecal coliform levels. A seasonal cycle could not be determined since strains of V. cholerae were detectable at low levels (ca. 1 to 10 cells/liter) throughout the year. Although none of the Chesapeake Bay isolates was agglutinable in V. cholerae O group 1 antiserum, the majority for Y-1 adrenal cells. Furthermore, rabbit ileal loop and mouse lethality tests were also positive for the Chesapeake Bay isolates, with average fluid accumulation in positive ileal loops ranging from 0.21 to 2.11 ml/cm. Serotypes of the strains of V. cholerae recovered from Chesapeake Bay were those of wide geographic distribution. It is concluded from the data assembled to date, that V. cholerae is an autochthonous estuarine bacterial species resident in Chesapeake Bay.     

Taxonomy of bacteria isolated from a coastal, marine fish-rearing unit

Phenetic data on almost 600 aerobic, heterotrophic bacteria from a marine fishrearing unit were collected and analysed using numerical taxonomic techniques. Reference strains, representing 42 taxa were included in the analyses. At similarity levels of 85% or above, with analyses prepared from the simple matching coefficient (S_SM), 81% of the isolates were recovered in eight major and 43 minor phena. Five of the major phena were equated with Acinetobacter calcoaceticus, Photobacterium phosphoreum and Vibrio spp. (three groups); the three unidentified phena contained Gram negative rods with polar flagella which were considered to be intermediate between Cytophaga/Flexibacter and Flavobacterium (two phena), and Gram variable rods. The surface of healthy turbot ( Scophthalmus maximus L. ) was populated by a diverse array of bacteria, including Alcaligenes faecalis, Bacillus firmus, Photobacterium angustum,'Photobacterium logei' and Pseudomonas fluorescens. Taxa, isolated as pure culture growth from within the lesions of moribund animals, included Alteromonas haloplanktis and unidentified Gram negative, budding bacteria. Vibrio anguillarum was not recovered from any turbot suspected of suffering from 'vibriosis'.     

Evidence for colonization and destruction of hinge ligaments in cultured juvenile Pacific oysters (Crassostrea gigas) by cytophaga-like bacteria.

Several strains of cytophaga-like gliding bacteria (CLB) were isolated as numerically dominant or codominant components of bacterial populations associated with proteinaceous hinge ligaments of cultured juvenile Pacific oysters, Crassostrea gigas. These bacteria were morphologically similar to long, flexible bacilli occurring within degenerative lesions in oyster hinge ligaments. Among bacteria isolated from hinge ligaments, only CLB strains were capable of sustained growth with hinge ligament matrix as the sole source of organic carbon and nitrogen. In vitro incubation of cuboidal portions of ligament resilium with ligament CLB resulted in bacterial proliferation on the surfaces and penetration deep into ligament matrices. Bacterial proliferation was accompanied by loss of resilium structural and mechanical integrity, including complete liquefaction, at incubation temperatures between 10 and 20 degrees C. The morphological, distributional, and degradative characteristics of CLB isolated from oyster hinge ligaments provide compelling, albeit indirect, evidence that CLB are the agents of a degenerative disease affecting juvenile cultured oysters. The motility, metabolic, and hydrolytic characteristics of hinge ligament CLB and the low moles percent G + C values (32.4 to 32.9) determined for three representative strains indicate that they are marine Cytophaga spp.     

Evidence for colonization and destruction of hinge ligaments in cultured juvenile Pacific oysters (Crassostrea gigas) by cytophaga-like bacteria

Several strains of cytophaga-like gliding bacteria (CLB) were isolated as numerically dominant or codominant components of bacterial populations associated with proteinaceous hinge ligaments of cultured juvenile Pacific oysters, Crassostrea gigas. These bacteria were morphologically similar to long, flexible bacilli occurring within degenerative lesions in oyster hinge ligaments. Among bacteria isolated from hinge ligaments, only CLB strains were capable of sustained growth with hinge ligament matrix as the sole source of organic carbon and nitrogen. In vitro incubation of cuboidal portions of ligament resilium with ligament CLB resulted in bacterial proliferation on the surfaces and penetration deep into ligament matrices. Bacterial proliferation was accompanied by loss of resilium structural and mechanical integrity, including complete liquefaction, at incubation temperatures between 10 and 20 degrees C. The morphological, distributional, and degradative characteristics of CLB isolated from oyster hinge ligaments provide compelling, albeit indirect, evidence that CLB are the agents of a degenerative disease affecting juvenile cultured oysters. The motility, metabolic, and hydrolytic characteristics of hinge ligament CLB and the low moles percent G + C values (32.4 to 32.9) determined for three representative strains indicate that they are marine Cytophaga spp.     

Effects of sudden temperature shifts on pure cultures of four strains of freshwater bacteria

Three psychrotrophic and one mesophilic strains were isolated from winter water samples of different freshwater biotopes and identified asCytophaga johnsonae (C-21),Cytophaga sp. (M-17),Pseudomonas fluorescens (KD), andEnterobacter cloacae (BS-2). Temperature shift-up experiments with emphasis on low temperatures were carried out with aerated pure batch cultures in glucose mineral medium. The effects of sudden temperature increases on growth rates and substrate conversion were investigated. All three psychrotrophic strains in the temperature increase experiments at low temperatures showed differing reactions within the linear zone of the Arrhenius plot. TheC. johnsonae (C-21) shift-up cultures adjusted the growth rate immediately to the rate of the temperature adapted cultures, whereasCytophaga sp. (M-17) shift-up cultures showed a lower andP. fluorescens (KD) a higher growth rate. The mesophilicE. cloacae (BS-2), likeC. johnsonae (C-21), adjusted immediately to the new growth rate. Substrate conversion increased in all experiments immediately after the shift-up. The extracellular substrate conversion byP. fluorescens (KD) of glucose to gluconate and 2-ketogluconate was particularly affected by the sudden temperature increase.     

Effects of temperature,ph, salinity,and inorganic nitrogen on the rate of ammonium oxidation by nitrifiers isolated from wetland environments

Ammonium-oxidizing bacteria were examined in two wetland environments, a freshwater marsh and an estuarine bay, during a 2-year period. Two predominant types were consistently isolated, one from each environment. Both isolates were identified as species ofNitrosomonas. Using a closed culture, high cell density assay, the effects of temperature, pH, salinity, Na⁺, K⁺, nitrite, nitrate, and ammonium concentrations on ammonium oxidation were determined. Maximum activity was observed for the freshwater isolate at 35°C, pH 8.5, salinities of 0.3 to 0.5% Na⁺ and K⁺, and ammonium concentrations greater than 0.5 g/l. For the estuarine isolate, maximum activity was observed at 40°C, pH 8.0, salinities of 0.5 to 1.0%, 1.0% Na⁺ and K⁺, and 0.2 g/l ammonium. The estuarine isolate had a Na⁺ requirement which could be partially substituted by the K⁺, suggesting that the organism is a true estuarine bacterium. Nitrite inhibited both isolates at concentrations greater than 5 mg/l, whereas nitrate had no significant effect on either isolate.     

Salinity tolerances of 62 strains of Pflesteria and Pfiesteria-like heterotrophic flagellates (Dinophyceae)

The salinity tolerance of 62 strains of Pfiesteria and Pfiesteria‐like heterotrophic dinoflagellates was measured. All strains were acclimated at 12 psu for at least 1 year before experimentation. Strains isolated from the Chesapeake Bay and Neuse River systems tolerated lower salinities than strains isolated from the Wilmington River system (P< 0.005). Swimming cells were still observed after 5 days at 0.5 psu for one strain, and at 1 psu for most other Chesapeake Bay and Neuse River strains. Swimming cells for the Wilmington River were still observed after 5 days at 3–5 psu, but no swimming cells were observed at ≤ 2 psu. With regard to the upper salinity tolerance, the Wilmington River strains tolerated higher salinities than the Chesapeake Bay and Neuse River systems (P< 0.005). Most Wilmington River strains were swimming after 5 days at salinities ≥ 50 psu, whereas the Chesapeake Bay and Neuse River system strains rarely had swimming cells at salinities exceeding 35–45 psu. For all three water systems and for both lower and higher salinities, cells apparently encysted in many instances. However, when salinities were returned to 12 psu, swimming cells often re‐appeared. Statistically significant geographic differences in salinity tolerance suggest a geographic adaptation has occurred and that salinity tolerance is under genetic control. The results also suggest there is diversity among the strains.     

Role of plasmids in mercury transformation by bacteria isolated from the aquatic environment

Eight mercury-resistant bacterial strains isolated from the Chesapeake Bay and one strain isolated from the Cayman Trench were examined for ability to volatilize mercury. Mercury volatilization was found to be variable in the strains tested. In addition, plasmids were detected in all strains. After curing, two of the bacterial strains lost mercury resistance, indicating that volatilization is plasmid mediated in these strains. Only two cultures demonstrated ability to methylate mercuric chloride under either aerobic or anaerobic conditions. Methylation of mercury, compared with volatilization, appears to be mediated by a separate genetic system in these bacteria. It is concluded that mercury volatilization in the estuarine environment can be mediated by genes carried on plasmids.     

Role of plasmids in mercury transformation by bacteria isolated from the aquatic environment.

Eight mercury-resistant bacterial strains isolated from the Chesapeake Bay and one strain isolated from the Cayman Trench were examined for ability to volatilize mercury. Mercury volatilization was found to be variable in the strains tested. In addition, plasmids were detected in all strains. After curing, two of the bacterial strains lost mercury resistance, indicating that volatilization is plasmid mediated in these strains. Only two cultures demonstrated ability to methylate mercuric chloride under either aerobic or anaerobic conditions. Methylation of mercury, compared with volatilization, appears to be mediated by a separate genetic system in these bacteria. It is concluded that mercury volatilization in the estuarine environment can be mediated by genes carried on plasmids.