Comparative proteomics and activity of a green sulfur bacterium through the water column of Lake Cadagno,Switzerland

Primary production in the meromictic Lake Cadagno, Switzerland, is dominated by anoxygenic photosynthesis. The green sulfur bacterium Chlorobium clathratiforme is the dominant phototrophic organism in the lake, comprising more than half of the bacterial population, and its biomass increases 3.8‐fold over the summer. Cells from four positions in the water column were used for comparative analysis of the Chl. clathratiforme proteome in order to investigate changes in protein composition in response to the chemical and physical gradient in their environment, with special focus on how the bacteria survive in the dark. Although metagenomic data are not available for Lake Cadagno, proteome analysis was possible based on the completely sequenced genome of an isolated strain of Chl. clathratiforme. Using LC‐MS/MS we identified 1321 Chl. clathratiforme proteins in Lake Cadagno and quantitatively compared 621 of these in the four samples. Our results showed that compared with cells obtained from the photic zone, cells collected from the dark part of the water column had the same expression level of key enzymes involved in carbon metabolism and photosynthetic light harvesting. However, most proteins participating in nitrogen and sulfur metabolism were twofold less abundant in the dark. From the proteome analysis we were able to show that Chl. clathratiforme in the photic zone contains enzymes for fixation of N₂ and the complete oxidation of sulfide to sulfate while these processes are probably not active in the dark. Instead we propose that Chl. clathratiforme cells in the dark part of the water column obtain energy for maintenance from the fermentation of polyglucose. Based on the observed protein compositions we have constructed possible pathways for C, N and S metabolism in Chl. clathratiforme.     

Long-Term Population Dynamics of Phototrophic Sulfur Bacteria in the Chemocline of Lake Cadagno, Switzerland

Population analyses in water samples obtained from the chemocline of crenogenic, meromictic Lake Cadagno, Switzerland, in October for the years 1994 to 2003 were studied using in situ hybridization with specific probes. During this 10-year period, large shifts in abundance between purple and green sulfur bacteria and among different populations were obtained. Purple sulfur bacteria were the numerically most prominent phototrophic sulfur bacteria in samples obtained from 1994 to 2001, when they represented between 70 and 95% of the phototrophic sulfur bacteria. All populations of purple sulfur bacteria showed large fluctuations in time with populations belonging to the genus Lamprocystis being numerically much more important than those of the genera Chromatium and Thiocystis. Green sulfur bacteria were initially represented by Chlorobium phaeobacteroides but were replaced by Chlorobium clathratiforme by the end of the study. C. clathratiforme was the only green sulfur bacterium detected during the last 2 years of the analysis, when a shift in dominance from purple sulfur bacteria to green sulfur bacteria was observed in the chemocline. At this time, numbers of purple sulfur bacteria had decreased and those of green sulfur bacteria increased by about 1 order of magnitude and C. clathratiforme represented about 95% of the phototrophic sulfur bacteria. This major change in community structure in the chemocline was accompanied by changes in profiles of turbidity and photosynthetically available radiation, as well as for sulfide concentrations and light intensity. Overall, these findings suggest that a disruption of the chemocline in 2000 may have altered environmental niches and populations in subsequent years.     

Pelodictyon phaeoclathratiforme sp. nov., a new brown-colored member of the Chlorobiaceae forming net-like colonies

A new strain of the green sulfur bacteria was isolated from the monimolimnion of Buchensee (near Radolfzell, Lake Constance region, FRG). Single cells were rod-shaped, nonmotile and contained gas vacuoles. Typical net-like colonies were formed by ternary fission of the cells. As photosynthetic pigments bacteriochlorophylls a, e, isorenieratene and -isorenieratene were present. Sulfide, sulfur and thiosulfate were used as electron donors during anaerobic phototrophic growth. Besides carbon dioxide, acetate and propionate could serve as carbon sources under mixotrophic conditions in the light. Like all other members of the green sulfur bacteria, the new bacterium is strictly anaerobic and obligately phototrophic. The possession of gas vacuoles and the formation of net-like colonies and the guanine plus cytosine content of the DNA (47.9 mol% G+C) are typical characteristics of the genus Pelodictyon. Because of its photosynthetic pigments which differ from those of Pelodictyon clathratiforme, strain BU 1 represents a new species, P. Phaeoclathratiforme sp. nov.     

Kluyveromyces lactis and Saccharomyces cerevisiae, two potent deacidifying and volatile-sulphur-aroma-producing microorganisms of the cheese ecosystem

Cheese flavour is the result of complex biochemical transformations attributed to bacteria and yeasts grown on the curd of smear-ripened cheeses. Volatile sulphur compounds (VSCs) are responsible for the characteristic aromatic notes of several cheeses. In the present study, we have assessed the ability of Kluyveromyces lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae strains, which are frequently isolated from smear-ripened cheeses, to grow and deacidify a cheese medium and generate VSCs resulting from l-methionine degradation. The Kluyveromyces strains produced a wider variety and higher amounts of VSCs than the S. cerevisiae ones. We have shown that the pathway is likely to be proceeding differently in these two yeast genera. The VSCs are mainly generated through the degradation of 4-methylthio-oxobutyric acid in the Kluyveromyces strains, in contrast to the S. cerevisiae ones which have higher l-methionine demethiolating activity, resulting in a direct conversion of l-methionine to methanethiol. The deacidification activity which is of major importance in the early stages of cheese-ripening was also compared in S. cerevisiae and Kluyveromyces strains.     

The rhizobia of lupinus densiflorus Benth., with some remarks on the classification of root nodule bacteria

Summary Four strains of rhizobia from Lupinus densiflorus Benth. were found to differ from the normal slow-growing strains of Rhizobium lupini by a rapid growth on agar medium, a somewhat different pattern of carbon metabolism, good growth in simple synthetic media, and also in their host plant relationships. Three strains had subpolar flagella like other lupine rhizobia, and the same was found to be predominant in a fourth strain previously described as having peritrichous flagellation.Two strains formed effectively nitrogen-fixing root nodules in Lotus corniculatus and Anthyllis vulneraria where the other two formed semieffective or ineffective nodules. All four strains formed ineffective nodules in Lotus uliginosus and Ornithopus sativus. The slow-growing strains of Rh. lupini mostly produce ineffective nodules in Lotus corniculatus but have now been seen to be effective in Lotus uliginosus.Instead of trying to define Rh. lupini as a cross-inoculation group it seems preferable to abandon it as a species and to transfer the fastgrowing strains to Rhizobium leguminosarum sensu Graham (1964) and De Ley and Rassel (1965), in spite of their predominantly subpolar flagellation. The familiar slow-growing strains would remain in the broad group of slow-growing root nodule bacteria with purely subpolar flagellation, called Phytomyxa japonica by Graham (1964) and Rhizobium japonicum by De Ley and Rassel (1965).     

A serological examination of some acetic acid bacteria

Summary Antigenic studies of twenty-five strains of acetic acid bacteria show that non-overoxidising strains form a serologically distinct group which by other characteristics can be identified asAcetobacter suboxydans (Bergey's Manual, 7th Ed.) The overoxidising strains do not constitute a serologically homogeneous group although evidence is presented that interrelationships can be established between certain strains.     

Vorkommen und Leistungsfähigkeit von Hydrogenase bei einigen Grünalgen

Summary Hydrogenase was shown to be present in the green algae, Chlorella pyrenoidosa (two different Emerson strains), Chlorella vulgaris (Pratt strain), Coelastrum proboscideum var. dilatatum, and Selenastrum gracile. In three of the algae studied, maximum activity of hydrogenase is reached after about 4 hours of anaerobiosis. The adaptation of the other two strains, however, requires about 12 and 30 hours respectively. By contrast, Chlorella ellipsoidea (strain Marburg St), Hormidium flaccidum, and Stichococcus bacillaris do not develop hydrogenase activity during up to 100 hours of anaerobic incubation.     

Nitrogen fixation and photoproduction of molecular hydrogen by Thiorhodaceae

Summary Nitrogen fixation has been shown to be a characteristic of two previously untested strains of the purple sulfur bacteriumChromatium sp.Chromatium strains have been shown to produce molecular hydrogen when suppliedD-L malate and bicarbonate in the presence of light and the absence of exogenous ammonia and molecular nitrogen. These results are discussed in relation to current findings on the nitrogen metabolism of the photosynthetic bacteria. Supported in part by grants from the Rockefeller Foundation, the Atomic Energy Commission, and the Research Committee of the Graduate School from funds provided by the Wisconsin Alumni Research Foundation.     

DL-4-hydroxyphenylglycine catabolism in Pseudomonas putida MW27

Thirteen bacteria were isolated on D-4-hydroxyphenylglycine as sole carbon and energy source. Seven strains transaminated only the D-enantiomer while the other six isolates transaminated both enantiomers of 4-hydroxyphenylglycine. One of the six strains utilizing both enantiomers was characterized as a Pseudomonas putida. This strain, MW27, employed two enantioselective transaminases, to catalyze the initial step in the metabolism of DL-4-hydroxyphenylglycine. The product of the transamination, 4-hydroxyphenylglyoxylate, was further metabolized via 4-hydroxybenzaldehyde and 4-hydroxybenzoate to protocatechuate. Preliminary results indicate that both transaminases are co-ordinately synthesized together with the 4-hydroxyphenylglyoxylate decarboxylase and the NADP⁺-dependent 4-hydroxybenzaldehyde dehydrogenase.     

Metabolism of l-alanine in Desulfotomaculum ruminis and two marine Desulfovibrio strains

The degradation of l-alanine by three strains of sulfate-reducing bacteria that can grow with l-alanine as an energy source was investigated. In Desulfotomaculum ruminis and most likely also in two marine Desulfovibrio strains alanine is converted to pyruvate via an NAD-dependent alanine dehydrogenase. D. ruminis contained high activities of soluble NADH and NADPH dehydrogenases. In the marine strains the activities were much lower and the NADH dehydrogenase was partly associated with the membrane fraction.     

Taxonomic studies on some gram-positive coryneform hydrogen bacteria

Recently isolated coryneform hydrogen bacteria were investigated under taxonomical aspects. Strains 7 C, RH 10, and 14 g are characterized by the snapping type of cell division, 68.5 to 69.7% GC content, dl-diaminopimelic acid in the cell wall, content of metachromatic granules, weak utilization of sugars and inhibitory effect of citrate. The strains are placed to the group 1—genus Corynebacterium—of the classification of coryneform bacteria of Yamada and Komagata (1972) and the name Corynebacterium autotrophicum sp.nov. is proposed.Strains 11 X and RH 12 are characterized by the bending type of cell division, a GC content of 70.2 and 70.5%, ll-diaminopimelic acid in the cell wall, absence of metachromatic granules, utilization of several sugars and no changes in cell morphology by citrate. The strains have to be placed to group 6 of coryneform bacteria.     

ATP-linked citrate lyase activity in the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum

Cell-free extracts of the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum strains 1C and L have been shown to cleave citrate with the formation of oxaloacetate and acetyl-CoA. This capacity was found in autotrophically grown cells as well as in the cells grown on media with acetate or L-glutamate. Citrate lyase activity in cell-free extracts is only measurable in the presence of citrate, adenosine-5-triphosphate, coenzyme A and Mg²⁺ or Mn²⁺. It is concluded on the basis of the obtained data that C. limicola f. thiosulfatophilum contains adenosine-5-triphosphate-linked citrate lyase (E.C.4.1.3.8). In contrast to green bacteria in the purple bacteria Ectothiorhodospira shaposhnikovii, Rhodospirillum rubrum and Thiocapsa roseopersicina citrate lyase activity was not found.     

Étude de quelques bacilles homolactiques isolés de vins

Résumé Les auteurs ont étudié 750 bactéries lactiques isolées de vins, en utilisant les tests et le système de classification de Rogosa et Sharpe. Parmi ce grand nombre de souches vingt-trois appartiennent au groupe des bacilles homolactiques et font l'objet du présent travail. Elles se répartissent de la façon suivante: 9 souches de Lactobacillus plantarum, 2 souches de Lactobacillus casei var. casei, 4 souches de Lactobacillus casei var. alactosus et 8 souches de Streptobacterium non classées, différentes des espèces précédentes.Les auteurs discutent la valcur de cette classification, lorsqu'on se place au point de vue technologique. Ils montrent qu'elle s'applique mal aux bactéries lactiques isolées de milieux fermentés acides comme le vin. Elle a peu d'intérêt pratique, car elle ne permet pas de repérer une souche et de prévoir par sa position systématique les constituants du vin que cette souche est susceptible de métaboliser.

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A study of some homofermentative lactic acid bacteria isolated from wines

Summary The authors have studied 750 strains of lactic acid bacteria isolated from wines. In this study the test and classification of Rogosa and Sharpe were used. Of the strains mentioned 23 belonged to the homolactie bacteria, including 9 strains of Lactobacillus plantarum, 2 strains of Lactobacillus casei var. casei, 4 strains of Lactobacillus casei var. alactosus, and 8 strains of a non-identified Streptobacterium species.The authors discuss the value of the classification mentioned from the point of view of wine technology. They conclude that it cannot be applied in the case of lactic acid bacteria isolated from acid fermentation products such as wine. It is only of little practical interest because it does not render the identification of the strains possible, nor does it permit a prediction of the wine constituents which the strains concerned are able to metabolize.

     

Oxidation of short-chain fatty acids by sulfate-reducing bacteria in freshwater and in marine sediments

Colony counts of acetate-, propionate- and l-lactate-oxidizing sulfate-reducing bacteria in marine sediments were made. The vertical distribution of these organisms were equal for the three types considered. The highest numbers were found just beneath the border of aerobic and anaerobic layers.Anaerobic mineralization of acetate, propionate and l-lactate was studied in the presence and in the absence of sulfate. In freshwater and in marine sediments, acetate and propionate were oxidized completely with concomitant reduction of sulfate. l-Lactate was always fermented. Lactate-oxidizing, sulfate-reducing bacteria, belonging to the species Desulfovibrio desulfuricans, and lactate-fermenting bacteria were found in approximately equal amounts in the sediments. Acetate-oxidizing, sulfate-reducing bacteria could only be isolated from marine sediments, they belonged to the genus Desulfobacter and oxidized only acetate and ethanol by sulfate reduction. Propionate-oxidizing, sulfate-reducing bacteria belonged to the genus Desulfobulbus. They were isolated from freshwater as well as from marine sediments and showed a relatively large range of usable substrates: hydrogen, formate, propionate, l-lactate and ethanol were oxidized with concomitant sulfate reduction. l-Lactate and pyruvate could be fermented by most of the isolated strains.     

The nickel resistance determinant cloned from the enterobacterium Klebsiella oxytoca: conjugational transfer,expression, regulation and DNA homologies to various nickel-resistant bacteria

Klebsiella oxytoca strain CCUG 15788, isolated from a mineral oil emulsion tank in Göteborg, Sweden, was found to be nickel-resistant (tolerating 10 mm NiCl₂ in non-complexing mineral-gluconate media; inducible resistance). The nickel resistance determinants were transferred by helper-assisted conjugation to various strains of Escherichia coli and Citrobacter freundii and expressed to between 5 and 10 mm NiCl₂. A 4.3 kb HindIII fragment was cloned from the genomic DNA of K. oxytoca. Ligated into the vector pSUP202, the fragment caused constitutive nickel resistance (of up to 3 or 10 mm Ni²⁺) in various E. coli strains. After cloning into the broad host range vector pVDZ'2 the fragment even expressed low nickel resistance in the transconjugant of Alcaligenes eutrophus AE104. With the 4.3 kb HindIII fragment as a biotinylated DNA probe it was shown by DNA-DNA hybridization that the nickel resistance determinant resides on the chromosome of K. oxytoca and not on its circular plasmid pKO1 (160 kb) or linear plasmid pKO2 (50 kb). Nickel resistance strongly correlated with the presence of the 4.3 kb HindIII fragment in the transconjugants. No homologies were detected when the nickel resistance determinants of other well-known nickel-resistant bacteria, such as A. eutrophus CH34 or A. denitrificans 4a-2, were used as target DNA. Among the 60 strains examined, positive signals only appeared with the 3.1 kb DNA fragment from A. xylosoxydans 31A and the genomic DNA of two enterobacterial strains (5-1 and 5–5) isolated from nickel-rich soil in New Caledonia.     

Variation in alpha-L-fucosidase properties among 28 inbred mouse strains: Six strains have high enzyme activity and heat-stabile enzyme with a variant pH-activity curve; twenty-two strains have low activity and heat-labile enzyme

Alpha-L-fucosidase in tissues of 28 inbred mouse strains varied with respect to three properties: high or low heat stability, a pH-activity curve with high or low relative activity at pH 2.8, and high or low activity. Alpha-L-fucosidase from six strains (A/J, BDP/J, LP/J, P/J, SEA/GNJ, and 129/J) had high heat stability, high pH 2.8 relative activity, and high activity, whereas the other 22 strains all had low heat stability, low pH 2.8 relative activity, and low activity. The heat-stability difference was seen in all organs tested (brain, liver, kidney, spleen, heart, skeletal muscle, lung, and testis) for two heat-stabile strains (P/J and 129/J) and four heat-labile strains (C57 BL/6J, C3H/HeJ, DBA/2J, and BALB/cJ) studied in detail. The findings suggested that two structural variants of alpha-L-fucosidase, probably genetically determined, exist in these 28 inbred mouse strains, although the presence of linkage disequilibrium between alleles of tightly linked structural and regulatory genes could not be excluded.This work was supported by grants from the National Institutes of Health (NS-15281 and NS-11766), the Muscular Dystrophy Association (H. Houston Merritt Clinical Center for Muscular Dystrophy and Related Diseases), the March of Dimes Birth Defects Foundation, and a generous gift from the Alexander Rapaport Foundation.     

Amino Acid Deamination by Ruminal <Emphasis Type="Italic">Megasphaera elsdenii</Emphasis> Strains

When ruminal fluid from a cow fed timothy hay was serially diluted (10-fold increments into anaerobic broth containing 15 mg ml⁻¹ Trypticase), the low dilutions (≤10⁻⁶) had optical densities greater than 2.0 and ammonia concentrations greater than 100 mM. The optical densities and ammonia concentrations of the 10⁻⁸ and 10⁻⁹ dilutions were very low, but large cocci were observed in the 10⁻⁸ dilution. The large cocci were isolated and identified by 16S rDNA sequencing as Megasphaera elsdenii. The freshly isolated strain (JL1) grew well on Trypticase, but less than 4% of the amino acid nitrogen in Trypticase was converted to ammonia. Optical density and ammonia production were twice as great if Casamino acids were provided, and similar results were obtained with seven other strains (B159, AW106, YT91, LC1, T81, J1, and YZ70). Specific activities of deamination (based on Casamino acids) of the eight strains ranged from 100 (strain JL1) to 325 (strain B159) nmol mg protein⁻¹ min⁻¹. None of the strains could utilize branched-chain amino acids as an energy source for growth, but specific activities of branched-chain amino acid deamination ranged from 15 to 65 nmol mg protein⁻¹ min⁻¹. All eight of the M. elsdenii strains grew well in the presence of 5 μM monensin, and only two of the strains were strongly inhibited by 20 μM monensin. On the basis of these results, it appears that M. elsdenii is deficient in peptidase activity and can utilize only a few amino acids. Some M. elsdenii strains produced ammonia and branched-chain volatile fatty acids nearly as fast as obligate amino acid-fermenting ruminal bacteria, but the extent of this production was at least fourfold lower. Because all of the strains could tolerate 5 μM monensin, it is unlikely that this feed additive would significantly inhibit M. elsdenii in vivo. Received: 12 December 2001 / Accepted: 5 February 2002     

Use of a polymerase-chain-reaction-amplified DNA probe from Pseudomonas putida to detect d-hydantoinase-producing microorganisms by direct colony hybridization

Pseudomonas putida strain DSM 84 produces N-carbamyl-d-amino acids from the corresponding d-5-monosubstituted hydantoins. The sequence of the d-hydantoinase gene from this strain (GenBank accession number L24157) was used to develop a DNA probe of 122 base pairs (bp) that could detect d-hydantoinase genes in other bacterial genera by DNA and by colony hybridization. Under conditions tolerating 32% mismatch, the probe was specific for all strains that expressed d-hydantoinase activity. These include Pseudomonadaceae of all rRNA groups, and bacteria belonging to the genera Agrobacterium, Serratia, Corynebacterium, and Arthrobacter. Environmental sampling was simulated by screening a mixture of unknown microorganims from commercial inocula for the biodegradation of industrial, municipal and domestic wastes. The 122-bp probe was specific for microorganisms that subsequently demonstrated d-hydantoinase activity. Bacterial species from four different genera were detected, which were Pseudomonas, Klebsiella, Enterobacter, and Enterococcus.     

Two new species of arthrobacter requiring respectively vitamin B12 and the terregens factor

Summary The morphological, cultural, nutritional and general physiological properties of two species of bacteria, isolated from soil, are described which require for growth vitamin B₁₂ and the terregens factor respectively. Both organisms undergo the change in morphology from a rod stage in young culture to a coccoid stage in older culture which is characteristic of members of the genus Arthrobacter Conn and Dimmick and are considered to be most appropriately assigned to this genus. In view of their differences from other described species and from each other the organism for which vitamin B₁₂ is essential is named Arthrobacter duodecadis n. sp. and that requiring the terregens factor Arthrobacter flavescens n. sp.Herrn Professor Dr. Dr. h. c. A. Rippel zum 70. Geburtstag.Contribution No. 455, Bacteriology Division.