The Isolation and Some Characteristics of Photosynthetic Bacteria (Chromatiaceae and Chlorobiaceae) from Antarctic Marine Sediments

Sediment samples taken at depths from 5 to 50 m in Borge Bay, Signy Island, Antarctica yielded purple ( Chromatiaceae ) and green ( Chlorobiaceae ) phototrophic bacteria when inoculated into Winogradsky columns or directly into Pfennig's medium. Pure cultures of Thiocapsa roseopersicina (2 strains), Thiocapsa pfennigii, Chromatium gracile, Chromatium vinosum, Thiocystis violaceae, Chlorobium limicola, Chlorobium vibrioforme and Prostaecochloris aestuarii were subsequently isolated from these enrichments. Thiocapsa roseopersicina and Chromatium vinosum were the species isolated most commonly. None of the isolates showed evidence of temperature adaptation; optimum growth was achieved between 25–30°C but all grew readily at low white light levels (spectral irradiance 160 μW/cm²) with mean generation times ranging from 8·9 to 14·2 h at 23°C depending upon the isolate. The green phototrophs grew readily in blue light (440–510 nm) of low irradiance (126 μW/cm²) which penetrates to a depth of 40–50 m in coastal waters surrounding Signy Island. Under these conditions Chlorobium limicola and Chlorobium vibrioforme had mean generation times of 22 and 12·2 h respectively compared with 10·3 and 8·9 when grown in white light of similar total irradiance. None of the purple phototrophs grew under blue light but they grew under yellow light (peak transmission 590 nm, irradiance 137 μW/cm²). Chromatium gracile and Thiocapsa roseopersicina had mean generation times of 22·3 and 18·3 h respectively compared with 14·2 and 12·3 h in white light of similar total irradiance.     

A thermophilic green sulfur bacterium from New Zealand hot springs,Chlorobium tepidum sp. nov.

Thermophilic green sulfur bacteria of the genus Chlorobium were isolated from certain acidic high sulfide New Zealand hot springs. Cells were Gram-negative nonmotile rods of variable length and contained bacteriochlorophyll c and chlorosomes. Cultures of thermophilic chlorobia grew only under anaerobic, phototrophic conditions, either photoautotrophically or photoheterotrophically. The optimum growth temperature for the strains of thermophilic green sulfur bacteria isolated was 47–48°C with generation times of about 2 h being observed. The upper temperature limit for growth was about 52°C. Thiosulfate was a major electron donor for photoautotrophic growth while sulfide alone was only poorly used. N₂ fixation was observed at 48°C and cell suspensions readily reduced acetylene to ethylene. The G+C content of DNA from strains of thermophilic chlorobia was 56.5–58.2 mol% and the organisms positioned phylogenetically within the green sulfur bacterial branch of the domain Bacteria. The new phototrophs are described as a new species of the genus Chlorobium, Chlorobium tepidum.This paper is dedicated to Professor Norbert Pfennig on the occasion of his 65th birthday     

Distribution and orientation of hydrogenase in various photosynthetic bacteria

All of the tested photosynthetic bacteria possessed hydrogenase activity. Cell membranes of representatives of the Chromatiaceae, Rhodospirillaceae, and Chlorobiaceae families were found to be impermeable to the oxidized redox dyes methyl viologen and benzyl viologen, whereas the reduced forms could easily penetrate the membranes. This permeability difference made possible the localization of the hydrogenase enzyme. Members of the Chromatiaceae and the Rhodospirillaceae contained a predominantly or exclusively membranebound enzyme. In contrast, the majority of hydrogenase activity was in the cytoplasm ofChlorobium limicola f. thiosulfatophilum. Hydrogenase, or at least its active center, was oriented toward the outer surface of the cell membrane in purple bacteria.     

Chemotherapeutic sensitivity and viability of Nocardia asteroides and Nocardia brasiliensis in drugs

Procedures for testing drug sensitivity ofNocardia were standardized.Five strains each ofN. asteroides (NA) andN. brasiliensis (NB) isolated from pathogenic materials were tested for drug sensitivity in Sabouraud's glucose agar (SGA), Sabouraud's glucose broth (SGB) and SGB with addition of 10 % horse serum against sulphadiazine (S), penicillin (P), streptomycin (St), chloramphenicol (C), tetracycline (T) and 4-4-diamino diphenyl sulphone (DDS). The viability of strains at minimum inhibitory concentrations (MIC) of different drugs were also tested.Results showed that liquid medium is preferable to solid medium for drug testing as pellicle formation observed on the surface of liquid medium helps in accurate assessment of MIC. Serum addition did not appear to be necessary. Ten days incubation at 37 °C gives optimum results for determination of MIC. Taking the maximum drug concentration attainable in blood during therapy, the strains were classified as sensitive and resistant. All strains were resistant to DDS and P. One strain of NB was sensitive to St. One strain each of NA and NB was borderline sensitive to C. Four out of 5 strains in both species were sensitive to S. All strains were sensitive to T. The organisms remained viable in drugs, thus suggesting the limitations of antinocardial therapy.From Mycology Research Unit, Department of Dermatology, Seth G.S. Medical College and K.E.M. Hospital, Bombay-12.Hon. Professor Dermato-Venereology.Research Fellow.     

PRODUCTION OF AXENIC CULTURES OF MICROMONAS PUSILLA (PRASINOPHYCEAE) USING ANTIBIOTIC 1

Bacteria-free cultures of the prasinophyte Micromonas pusilla (Butcher) Monton and Parke, UTEX LB 991, were produced by intially determining the effects of several antibiotics on the growth of this alga and then using a combination of these antibiotics to eliminate associated bacteria, Micrononas pusilla was resistant ot penicillin G, neomycin, gentamicin and streptomycin at bactericidal concentrations but sensitive to chloramphenicol and polymixin B. Passage of M. pusilla through the sequence of antibioties penicillin G → neomycin → gentamician → kanamycin resulted in an axenic culture of M, pusilla this method should be suitable for producing axenic culture of other strains of M. pusilla.     

Anoxygenic phototrophic bacterial community of Lake Shira (Khakassia)

The anoxygenic phototrophic bacterial community of the brackish meromictic Lake Shira (Khakassia) was investigated in August 2001, July 2002, and February-March 2003. In all the periods of investigation, the prevailing microorganisms were purple sulfur bacteria similar to Lamprocystis purpurea in morphology and pigment composition. Their highest number (3 x 10(5) cells/ml) was recorded in July 2002 at the depth of 15 m. According to 16S rRNA gene analysis, the strain of purple sulfur bacteria isolated in 2001 and designated ShAm01 exhibited 98.6% similarity to the type strain of Thiocapsa roseopersicina and 94.4-97.1% similarity to the type strains of Tca. pendens, Tca. litoralis, and Tea. rosea. The minor microorganisms of the anoxygenic phototrophic bacterial community within the period of investigation were nonsulfur purple bacteria phylogenetically close to Rhodovulum strictum (98.3% similarity, strain ShRb01), Ahrensia kielensis (of 93.9% similarity, strain ShRb02), Rhodomicrobium vannieli (of 99.7% similarity, strain ShRmc01), and green sulfur bacteria, phylogenetically close to Chlorobium limicola (of 98.7% similarity, strain ShCl03).     

Sepiapterin reductases from Chlorobium tepidum and Chlorobium limicola catalyze the synthesis of L-threo-tetrahydrobiopterin from 6-pyruvoyltetrahydropterin

The ORF sequences of the gene encoding sepiapterin reductase were cloned from the genomic DNAs of Chlorobium tepidum and Chlorobium limicola, which are known to produce L-threo- and L-erythro-tetrahydrobiopterin (BH4)-N-acetylglucosamine, respectively. The deduced amino acid sequence of C. limicola consists of 241 residues, while C. tepidum SR has three residues more at the C-terminal. The overall protein sequence identity was 87.7%. Both recombinant proteins generated from Escherichia coli were identified to catalyze reduction of diketo compound 6-pyruvoyltetrahydropterin to L-threo-BH4. This result suggests that C. limicola needs an additional enzyme for L-erythro-BH4 synthesis to yield its glycoside. The catalytic activity of Chlorobium SRs also supports the previously proposed mechanism of two consecutive reductions of C1' carbonyl group of 6-pyruvoyltetrahydropterin via isomerization reaction.     

Characterization of ammonium and methylamine transport systems in phototrophic sulfur bacteria

Abstract Transport of ammonium and methylamine into the cells of green sulfur bacterium Chlorobium limicola and purple sulfur bacterium Thiocapsa roseopersicina is carried out by a common transport system. This system has (for C. limicola and T. roseopersicina , respectively) pH optimum 7.0 and 7.5; V _max 0.6 and 4.2 nmol min⁻¹ (mg protein)⁻¹; K_m 5.9 × 10⁻⁵ M and 1.3 × 10⁻⁵ M, and is capable of forming 120- and 600-fold methylamine gradients. The methylamine transport can be energized by the artificially imposed transmembrane K⁺ diffusive potential and is inhibited by tetraphenylphosphonium or valinomycin and K⁺. The data presented indicate that methylamine transport in both studied species is exclusively driven by the membrane potential gradient (ΔΨ).     

Laminated microbial ecosystems on sheltered beaches in Scapa Flow, Orkney Islands

Abstract Laminated microbial sediment ecosystems which develop in the upper tidal zone of Scapa Flow beaches, Orkney Islands were investigated with respect to depth profiles of chlorophyll a , bacteriochlorophyll a , pH, redox, oxygen and the following inorganic sulfur compounds: free sulfide, FeS, polysulfides, polythionates, elemental sulfur and thiosulfate. In addition, particle size distribution and light penetration were determined at all sampling locations.
Three main types of laminated sediment ecosystems were recognized, designated the 'classical' type (layer of cyanobacteria underlain by layer of purple sulfur bacteria), the 'single-layer' type (chlorophyll a containing organisms absent, purple sulfur bacteria at sediment surface), and the 'inverted' type (chlorophyll a containing organisms underlying purple sulfur bacteria). The dominant purple sulfur bacterium was Thiocapsa roseopersicina and Chromatium vinosum was observed less commonly. The principal cyanobacterium found in these sulfureta was Oscillatoria sp.
The depth horizon at which maximum populations of purple sulfur bacteria were recorded often did not coincide with the sulfide/oxygen interface but was located closer to the sediment surface where polysulfides, polythionates, elemental sulfur and occasionally thiosulfate were present. The structure of these sulfureta is discussed in relation to the chemolithotrophic growth capacities of Thiocapsa in the presence of oxygen.     

Characterization of ATP citrate lyase from Chlorobium limicola.

ATP citrate lyase (EC 4.1.3.8) from Chlorobium limicola was partially purified. It was established that the consumption of substrates and the formation of products proceeded stoichiometrically and that citrate cleavage was of the si-type. ADP and oxaloacetate inhibited enzyme activity. Oxaloacetate also inhibited the growth of C. limicola.     

Growth yields of green sulfur bacteria in mixed cultures with sulfur and sulfate reducing bacteria

1. Dry weight yields from mixed cultures ofProsthecochloris aestuarii orChlorobium limicola with the sulfur reducingDesulfuromonas acetoxidans were determined on different growth limiting amounts of acetate, ethanol or propanol. The obtained yields agreed well with values predicted from stoichiometric calculations. 2. From mixed cultures of twoChlorobium limicola strains withDesulfovibrio desulfuricans orD. gigas on ethanol as the growth limiting substrate, dry weight yields were obtained as calculated for the complete utilization of the ethanol by the mixed cultures. 3. Dry weight yield determinations for two pure cultures ofChlorobium limicola with different growth limiting amounts of sulfide in the absence and presence of excess acetate confirmed that acetate is incorporated byChlorobium in a fixed proportion to sulfide; compared to the yield in the absence of acetate the yield is increased two to threefold in the presence of acetate. 4. The lowest possible sulfide concentrations necessary for optimal growth of mixed cultures of eitherProsthecochloris orChlorobium withDesulfuromonas on acetate were 7–8 mg H₂S per liter of medium. 5. Doubling times at the growth rate limiting light intensities of 5, 10, 20, 50, 100 and 200 lux were determined under optimal growth conditions for the following phototrophic bacteria:Prosthecochloris aestuarii, Chlorobium phaeovibriodes, Chromatium vinosum andRhodopseudomonas capsulata. Reasonably good growth was still obtained withProsthecochloris at 10 and 5 lux light intensity at which no growth of the purple bacteria could be observed.     

Evaluation of Culture Techniques for Isolation of Pseudomonas pseudomallei from Soil

Three selective enrichment broths and four selective agar media were evaluated for their ability to support the growth of Pseudomonas pseudomallei both at 35°C and at ambient temperature (range, 20 to 32°C; mean, 25°C). Colony counts of 50 strains of P. pseudomallei and recovery studies with 1 soil strain in 60 simulated soil samples demonstrated that enrichment with Trypticase soy broth incorporating 5 mg of crystal violet per liter and 20 mg of colistin per liter (CVCB) and subculture to Ashdown medium supported the growth of all 50 strains and produced the highest recovery rates with the greatest suppression of other soil flora. An enrichment broth of MacConkey broth (purple) incorporating 10 mg of crystal violet per liter, 5 mg of bromcresol purple per liter, 25 mg of gentamicin per liter, and 650 mg of streptomycin per liter showed greater suppression of soil bacteria than CVCB, but it failed to support the growth of three strains of P. pseudomallei. Recovery rates were essentially the same irrespective of whether the soil samples were incubated at 35°C or at ambient temperature, provided cultures were incubated in protected shade for an extended period. This is an important feature for field work in large-scale epidemiological surveys in which resources are limited.     

Obligately and facultatively autotrophic,sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts

A variety of autotrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria were isolated from thermogenic composts at temperatures of 60–80° C. All were penicillin G sensitive, which proves that they belong to the Bacteria domain. The obligately autotrophic, non-spore-forming strains were gram-negative rods growing at 60–80°C, with an optimum at 70–75°C, but only under microaerophilic conditions (5 kPa oxygen). These strains had similar DNA G+C content (34.7–37.6 mol%) and showed a high DNA:DNA homology (70–87%) with Hydrogenobacter strains isolated from geothermal areas. The facultatively autotrophic strains isolated from hot composts were gram-variable rods that formed spherical and terminal endospores, except for one strain. The strains grew at 55–75° C, with an optimum at 65–70° C. These bacteria were able to grow heterotrophically, or autotrophically with hydrogen; however, they oxidized thiosulfate under mixotrophic growth conditions (e.g. pyruvate or hydrogen plus thiosulfate). These strains had similar DNA G+C content (60–64 mol%) to and high DNA:DNA homology (> 75%) with the reference strain of Bacillus schlegelii. This is the first report of thermogenic composts as habitats of thermophilic sulfur- and hydrogen-oxidizing bacteria, which to date have been known only from geothermal manifestations. This contrasts with the generally held belief that thermogenic composts at temperatures above 60° C support only a very low diversity of obligatory heterotrophic thermophiles related to Bacillus stearothermophilus. Received: 20 July 1995 / Accepted: 25 September 1995     

Photooxidase activity of isolated chromatophores and intact cells of phototrophic bacteria

Illumination causes an uptake of oxygen by isolated chromatophores of purple and green bacteria incubated with electron donors. Photooxidase activity of Rhodospirillum rubrum, Chromatium minutissimum, Rhodopseudomonas sphaeroides and Thiocapsa roseopersicina chromatophores is sensitive, and photooxidase activity of Ectothiorhodospira shaposhnikovii and Chlorobium limicola f. thiosulfatophilum is resistant to o-phenanthroline. O₂ uptake by illuminated chromatophores of R. rubrum and C. limicola is stimulated upon the increase of pH of incubation mixture from 5 to 9. Photooxidase activity is also manifested in the intact bacterial cells and not merely in the isolated chromatophores. O₂ uptake by the illuminated R. rubrum cells treated with CN^- is stimulated by 2-heptyl-4-hydroxyquinoline-N-oxide and a protonophorous uncoupler. The interaction of the photosynthetic and respiratory systems of the electron transfer in the bacterial cells and the probable causes of the strong anaerobic way of life of the green sulfur bacteria are discussed.HQNO 2-heptyl-4-hydroxyquinoline-N-oxide - TMPD N,N,-N,N-tetramethyl-p-phenylenediamine     

Phylogeny of green sulfur bacteria on the basis of gene sequences of 16S rRNA and of the Fenna-Matthews-Olson protein

The phylogeny of green sulfur bacteria was studied on the basis of gene sequences of the 16S rRNA and of the Fenna-Matthews-Olson (FMO) protein. Representative and type strains (31 strains total) of most of the known species were analyzed. On the basis of fmoA gene sequences from Chlorobium tepidum ATCC 49652(T) and Chlorobium limicola DSM 249(T) available from the EMBL database, primers were constructed that allowed sequence analysis of a major part of the fmoAgene. The largely congruent phylogenetic relationship of sequences of the fmoA gene and of 16S rDNA gives considerable support to the phylogeny of green sulfur bacteria previously suggested on the basis of 16S rDNA sequences. Distinct groups of strains were recognized on the basis of 16S rDNA and FMO sequences and supported by characteristic signature amino acids of FMO. Marine strains formed clusters separate from freshwater strains. The resulting phylogenetic grouping and relationship of the green sulfur bacteria do not correlate with their current taxonomic classification.     

Anoxygenic phototrophic bacteria in eutrophic coastal lagoons of the French Mediterranean and Atlantic Coasts (Prévost Lagoon, Arcachon Bay, Certes fishponds)

Sediment samples collected from coastal lagoons on the French Mediterranean (Prévost Lagoon) and Atlantic coasts (Arcachon Bay and Certes fishponds) have been studied in order to determine the population densities and the species diversity of the different groups of anoxygenic phototrophic bacteria (purple sulfur bacteria, purple nonsulfur bacteria and green sulfur bacteria) present in these ecosystems. Several strains of each group were isolated in pure culture and characterized by their physiological properties. The occurrence of purple nonsulfur bacteria in organic rich sediments of the Arcachon Bay and the dominance of purple sulfur bacteria in the Prévost lagoon and Certes fishponds are discussed with respect to their community structure and abundance. The diversity differences of the phototrophic bacterial strains isolated from both environments are also discussed.     

Formation of poly(3-hydroxyalkanoates) by phototrophic and chemolithotrophic bacteria

The formation of poly(3-hydroxyalkanoic acid), PHA, by various strains of chemolithotrophic and phototrophic bacteria has been examined. Chemolithotrophic bacteria were grown aerobically under nitrogen-limiting conditions on various aliphatic organic acids. Phototrophic bacteria were grown anaerobically in the light on a nitrogen-rich medium and were subsequently transferred to a nitrogen-free medium containing acetate, propionate, valerate, heptanoate or octanoate as carbon source. All 41 strains investigated in this study were able to synthesize and accumulate PHA. All 11 strains of chemolithotrophic bacteria and all 15 strains belonging to the non-sulfur purple bacteria synthesized a polymer, which contained 3-hydroxy-valerate (3HV) beside 3-hydroxybutyrate (3HB), if the cells were cultivated in the presence of propionate, valerate or heptanoate. Many non-sulfur purple bacteria synthesized copolyesters of 3HB and 3HV even with acetate as carbon source. In contrast, most sulfur purple bacteria did not incorporate 3HV at all. Among 15 strains tested, only Chromatium vinosum strain 1611, C. purpuratum strain BN5500 and Lamprocystis roseopersicina strain 3112 were able to synthesize polyesters containing 3HV with propionate, valerate or heptanoate as carbon source.     

Multiple antibiotic resistance in Rhizobium japonicum.

A total of 48 strains of the soil bacterium Rhizobium japonicum were screened for their response to several widely used antibiotics. Over 60% of the strains were resistant to chloramphenicol, polymyxin B, and erythromycin, and 47% or more of the strains were resistant to neomycin and penicillin G, when tested by disk assay procedures. The most common grouping of resistances in strains was simultaneous resistance to tetracycline, penicillin G, neomycin, chloramphenicol, and streptomycin (25% of all strains tested). The occurrence of multiple drug resistance in a soil bacterium that is not a vertebrate pathogen suggests that chemotherapeutic use of antibiotics is not required for the development of multiple drug resistance.     

Multiple antibiotic resistance in Rhizobium japonicum.

A total of 48 strains of the soil bacterium Rhizobium japonicum were screened for their response to several widely used antibiotics. Over 60% of the strains were resistant to chloramphenicol, polymyxin B, and erythromycin, and 47% or more of the strains were resistant to neomycin and penicillin G, when tested by disk assay procedures. The most common grouping of resistances in strains was simultaneous resistance to tetracycline, penicillin G, neomycin, chloramphenicol, and streptomycin (25% of all strains tested). The occurrence of multiple drug resistance in a soil bacterium that is not a vertebrate pathogen suggests that chemotherapeutic use of antibiotics is not required for the development of multiple drug resistance.     

Synthesis and antibacterial activity of 2,4,6-tri substituted s-triazines

Various 2,4,6-tri substituted s-triazines were synthesized and screened for antibacterial activity against Gram-positive and Gram-negative organisms. These s-triazine derivatives displayed high in vitro antibacterial activities comparable to penicillin and streptomycin against tested microorganisms.