Utilization of methanol by rhodospirillaceae

Enrichment culture of organisms growing anaerobically in the light in methanol-bicarbonate medium resulted in isolation of strains of Rhodopseudomonas gelatinosa and Rhodopseudomonas acidophila. The pH optimum for growth on methanol for all strains tested was approximately one unit higher than for growth on carbon sources containing more than one carbon atom. At the appropriate pH, 17 strains of Rhodospirillaceae out of 39 in a culture collection grew anaerobically in the light on methanol-bicarbonate. Rhodopseudomonas acidophila strain 10050 showed the most abundant growth and was studied in more detail. Its growth on methanol was stimulated by yeast extract or vitamin-free casamino acids. The organism grew on methanol-bicarbonate, methanol-formate or formate alone as the sole carbon sources. No growth was observed on methylamine or formaldehyde. In the presence of excess bicarbonate a maximum yield of 98 g cell material from 100 g methanol was obtained. Ribulose diphosphate carboxylase was present in the methanol-bicarbonate-grown organism at six times the specific activity of that in the succinate-grown organism.     

Anaerobic Growth of Candida albicans Does Not Support Biofilm Formation Under Similar Conditions Used for Aerobic Biofilm

C. albicans is an opportunistic fungus causing life-threatening systemic infections particularly in immunocompromised individuals. The organism is a commensal in humans and grows either aerobically, e.g., the oral cavity, or anaerobically, e.g., the gut. We studied anaerobic growth of C. albicans in a defined yeast nitrogen base dextrose medium after adaptation and subculturing in an anaerobic chamber. At 37°C in suspension culture, much slower growth was observed anaerobically with a generation time of 248 min compared to 98 min for aerobic growth. Although the organism grew well on solid medium, shaking increased the growth rate in suspension culture at 37°C. Growth was enhanced at acidic pH compared to neutral or alkaline pH. Cells grown anaerobically produced hyphae, but did not produce biofilm on plastic surface or denture acrylic under either static conditions or with mild shaking, conditions that support aerobic biofilm formation.     

Mutant Strains of Rhodopseudomonas spheroides Lacking δ-Aminolevulinate Synthase: Growth, Heme, and Bacteriochlorophyll Synthesis

Two mutant strains of Rhodopseudomonas spheroides were described which lacked delta-aminolevulinate synthase activity. They required delta-aminolevulinate for growth; they did not respond to protoporphyrin or magnesium photoporphyrin, and only poorly to hemin. Synthesis of cytochromes and heme by mutant H-4 was dependent upon delta-aminolevulinate; this strain did not form bacteriochlorophyll either with or without delta-aminolevulinate and, consequently, grew only under aerobic conditions. Mutant H-5 formed bacteriochlorophyll in response to delta-aminolevulinate and grew both anaerobically in the light and aerobically in the dark; the amount of delta-aminolevulinate needed for optimal anaerobic growth was higher than that required aerobically. Synthesis of bacteriochlorophyll and heme by suspensions of mutant H-5 incubated anaerobically in the light was dependent upon delta-aminolevulinate; bacteriochlorophyll production was completely inhibited by high aeration and by puromycin. The mutants differed in their ability to take up radioactive delta-aminolevulinate from the external environment; mutant H-5 was less active than mutant H-4 or the wild type. It was suggested that R. spheroides made only one form of delta-aminolevulinate synthase, which provided delta-aminolevulinate for bacteriochlorophyll and heme synthesis.     

沼泽红假单胞菌培养条件的初步研究

就沼泽红假单胞菌Ｐ３．９菌株的主要培养条件进行了初步研究。Ｐ３．９菌株在厌氧光照条件下能利用多种类型的有机碳源物质;其细胞生长的最适ｐＨ为６．５—８,最佳光照强度为２０００—３０００Ｌｘ;培养基中添加酵母膏能明显促进细胞生长。在适宜的培养条件下,菌液细胞数可高达４８亿个／ｍｌ。     

Formaldehyde incorporation by a new methylotroph (L3)

A number of bacterial strains have been isolated and investigated in our search for a promising organism in the production of single-cell protein from methanol. Strain L3 among these isolates was identified as an obligate methylotroph which grew only on methanol and formaldehyde as the sole sources of carbon and energy. The organism also grew well in batch and chemostat mixed-substrate cultures containing methanol, formaldehyde, and formate. Although formate was not utilized as a sole carbon and energy source, it was readily taken up and oxidized by either formaldehyde- or methanol-grown cells. The organism incorporated carbon by means of the ribulose monophosphate pathway when growing on either methanol, formaldehyde, or various mixtures of C1 compounds. Its C1-oxidation enzymes included phenazine methosulfate-linked methanol and formaldehyde dehydrogenase and a nicotinamide adenine dinucleotide-linked formate dehydrogenase. Identical inhibition by formaldehyde of the first two dehydrogenases suggested that they are actually the same enzyme. The organism had a rapid growth rate, a high cell yield in the chemostat, a high protein content, and a favorable amino acid distribution for use as a source of single-cell protein. Of special interest was the ability of the organism to utilize formaldehyde via the ribulose monophosphate cycle.     

Anaerobic degradation of halogenated benzoic acids by photoheterotrophic bacteria

Abstract From light-exposed enrichment cultures containing benzoate and a mixture of chlorobenzoates, a pure culture was obtained able to grow with 3-chlorobenzoate (3-CBA) or 3-bromobenzoate (3-BrBA) as the sole growth substrate anaerobically in the light. The thus isolated organism is a photoheterotroph, designated isolate DCP3. It is preliminarily identified as a Rhodopseudomonas palustris strain. It differs from Rhodopseudomonas palustris WS17, the only other known photoheterotroph capable of using 3-CBA for growth, in its independence of benzoate for growth with 3-CBA and in its wider substrate range: if grown on 3-CBA, it can also use 2-CBA, 4-CBA or 3,5-CBA.     

Formaldehyde incorporation by a new methylotroph (L3).

A number of bacterial strains have been isolated and investigated in our search for a promising organism in the production of single-cell protein from methanol. Strain L3 among these isolates was identified as an obligate methylotroph which grew only on methanol and formaldehyde as the sole sources of carbon and energy. The organism also grew well in batch and chemostat mixed-substrate cultures containing methanol, formaldehyde, and formate. Although formate was not utilized as a sole carbon and energy source, it was readily taken up and oxidized by either formaldehyde- or methanol-grown cells. The organism incorporated carbon by means of the ribulose monophosphate pathway when growing on either methanol, formaldehyde, or various mixtures of C1 compounds. Its C1-oxidation enzymes included phenazine methosulfate-linked methanol and formaldehyde dehydrogenase and a nicotinamide adenine dinucleotide-linked formate dehydrogenase. Identical inhibition by formaldehyde of the first two dehydrogenases suggested that they are actually the same enzyme. The organism had a rapid growth rate, a high cell yield in the chemostat, a high protein content, and a favorable amino acid distribution for use as a source of single-cell protein. Of special interest was the ability of the organism to utilize formaldehyde via the ribulose monophosphate cycle.     

RESPONSES OF THE ACIDOPHILIC ALGA EUGLENA MUTABILIS (EUGLENOPHYCEAE) TO CARBON ENRICHMENT AT pH 31

A defined medium (MAM) simulating acid mine drainage waters was developed which supported reproducible growth rates of three axenic strains of Euglena mutabilis Schmitz. Growth responses to various pHs and carbon sources were examined under defined culture conditions. A lab strain and two 5eld isolates, tested over pH range 1.5-9.0, grew best under acidic conditions (pH < 5.5) with highest growth rates at pH 3-4. Photoauxotrophic growth rates of all strains at pH 3 were improved significantly over unstirred batch controls by bubbling with air and even more by enrichment with 5% CO₂ in air. These results confirmed inorganic carbon limitation in batch culture. Organic carbon substrates were tested as possible carbon supplements in batch culture at pH 3. None of the strains survived in the dark on any of the twenty organic sources added. In the light, the lab strain exhibited some photoheterotrophic growth potential on glucose, sucrose, ethanol, and amino acids but growth was inhibited by acetate. Field strains showed little or no growth improvement with any organic substrate addition. Under simultaneous enrichment with acetate and 5% CO₂ acetate continued to be inhibitory. Simultaneous enrichment with glucose and 5% CO₂ gave higher yields of the lab strain than with CO₂ alone but did not enhance growth of the field strain. We conclude that E. mutabilis is an acidophilic photoauxotroph which appears unable to use organic carbon supplements for growth even under conditions of carbon limitation.     

Molecular cloning of a coding sequence of Bordetella pertussis filamentous hemagglutinin gene

Abstract The halophilic phototrophic bacterium Rhodospirillum salexigens was tested for growth on a variety of organic and inorganic nitrogenous compounds as sole nitrogen sources. In media containing acetate as carbon source, the amino acids glutamate, proline, and aspartate supported good growth of R. salexigens ; several other amino acids or ammonia did not support growth. Attempts to grow R. salexigens on ammonia led to the discovery that this organism excretes a highly basic substance under certain nitrogen nutritional conditions which raises the pH above that supporting growth. Cultures of R. salexigens transferred to media containing both pyruvate and acetate as carbon sources grew on ammonia as sole nitrogen source and the culture pH did not rise. Dual substrate experiments showed that R. salexigens utilized glutamate in preference to ammonia when both were present at equimolar concentrations.     

Determinants in microbial colonization of the murine gastrointestinal tract: pH, temperature, and energy-yielding metabolism of Torulopsis pintolopesii.

Torulopsis pintolopesii is an indigenous yeast that colonizes the secreting epithelia in the stomachs of mice and rats. A wild-type strain of this microbe was isolated and identified. To attempt to learn characteristics of the yeast that are advantageous to it in colonizing its natural habitat in vivo, we examined some aspects of its nutrition and energy-yielding metabolism and some environmental conditions that influence its growth in vitro. The yeast appeared to be limited in the compounds it can utilize as carbon and nitrogen sources. It grew best at 37 degrees C and did not grow at 23 or 43 degrees C. It grew optimally at neutral pH but could grow aerobically at pH values as low as 2.0 and anaerobically at pH values as low as 3.4. As assessed by measurements of growth rates and yield coefficients, it grew better aerobically than anaerobically. When grown aerobically, it had a cyanide-sensitive system for taking up O(2) and tested positively for cytochrome c oxidase activity. A petite mutant strain isolated from the wild-type strain had a growth rate and yield coefficient when incubated aerobically that were essentially the same as those of the wild-type parent grown anaerobically. Likewise similar to the wild-type parent grown anaerobically, the petite strain, though incubated aerobically, did not take up O(2). Yeast-free mice associated with either the wild-type or the petite mutant strain were colonized at essentially the same rates and to similar final population levels by both strains. The yeast's capacity to respire may be of little advantage to it in its natural environment. By contrast, its abilities to grow best at 37 degrees C and to grow at low pH values are undoubtedly advantageous characteristics in this respect. The limitations in its carbon and nitrogen nutrition are difficult to evaluate as ecological factors in its colonization of the natural habitat.     

Microaerophilic growth and induction of the photosynthetic reaction center in Rhodopseudomonas viridis.

Rhodopseudomonas viridis was grown in liquid culture at 30 degrees C anaerobically in light (generation time, 13 h) and under microaerophilic growth conditions in the dark (generation time, 24 h). The bacterium could be cloned at the same temperature anaerobically in light (1 week) and aerobically in the dark (3 to 4 weeks) if oxygen was limited to 0.1%. Oxygen could not be replaced by dimethyl sulfoxide, potassium nitrate, or sodium nitrite as a terminal electron acceptor. No growth was observed anaerobically in darkness or in the light when air was present. A variety of additional carbon sources were used to supplement the standard succinate medium, but enhanced stationary-phase cell density was observed only with glucose. Conditions for induction of the photosynthetic reaction center upon the change from microaerophilic to phototrophic growth conditions were investigated and optimized for a mutant functionally defective in phototrophic growth. R. viridis consumed about 20-fold its cell volume of oxygen per hour during respiration. The MICs of ampicillin, kanamycin, streptomycin, tetracycline, 1-methyl-3-nitro-1-nitrosoguanidine, and terbutryn were determined.     

The Physiology of Astrephomene gubernaculifera1

SYNOPSIS. A comparative physiologic study of 4- and 7-chromosome strains of Astrephomene gubernaculifera was done. The vitamins p-aminobenzoic acid, nicotinamide, biotin, thiamin HCl and vitamin B₁₂ were tested for their ability to support growth. Only vitamin B₁₂ was required for active growth altho the presence of thiamin HCl was necessary to produce structurally typical colonies. Astrephomene will not grow in the absence of an exogenous source of carbon. Of the 36 carbon sources tested, only pyruvate, butyrate, succinate and acetate permitted active growth. Sodium acetate was the best. Strains grew within the initial pH range of 5.0–7.5. At pH values above 8.0 growth declined rapidly. When media were buffered with TES (N-tris [hydroxymethyl]methyl-2-aminoethane-sulfonic acid) at an initial pH of 6.8 growth was enhanced. The organism grew at 15–40 C. Growth in the dark was slightly less than that in the light.     

Role of pH, lactate, and anaerobiosis in controlling the growth of some fermentative Gram-negative bacteria on beef.

At 5 degrees C four strains of fermentative, gram-negative bacteria (Serratia liquefaciens, Yersinia enterocolitica, Enterobacter cloacae, and Aeromonas hydrophila) grew aerobically and anaerobically on adipose tissue removed from beef muscle of low pH (5.4 to 5.6). All four strains also grew aerobically and anaerobically on muscle tissue of high pH (6.0 to 6.3). However, none of the four grew anaerobically on beef muscle of low pH, and the aeromonad strain also failed to grow aerobically on such muscle. Growth of S. liquefaciens and E.cloacae on vacuum-packaged beef muscle was dependent on the pH of the tissue and the oxygen transmission rate of the packaging film. Although the four strains grew in broth buffered at pH 5.55, L-lactate, at the concentration found in muscle of low pH (ca. 100 mM), prevented anaerobic growth of all four isolates and prevented the aerobic growth of th aeromonad. At pH 6.1 in buffered broth, the concentration of L-lactate occurring in muscle of high pH did not prevent aerobic or anaerobic growth of any of the strains.     

The dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila. Comparison with dye-linked methanol dehydrogenases.

1. A dye-linked alcohol dehydrogenase was purified 20-fold from extracts of Rhodopseudomonas acidophila 10050 grown anaerobically in the light on methanol/HCO3-. 2. The enzyme resembled many previously reported methanol dehydrogenases from other methylotrophic organisms in coupling to phenazine methosulphate, requiring ammonia as an activator, possessing a pH optimum of 9 and a mol.wt. of approx. 116000. In many other respects the enzyme showed singular properties. 3. The stability of the enzyme under various conditions of temperature and pH was studied. 4. Primary aliphatic amines containing up to nine carbon atoms (the longest tested) were better activators than ammonia. 5. A wide range of primary alcohols and aldehydes served as substrates, with apparent Km values ranging from 57 mM for methanol to 6 micron for ethanol. 6. O2 was an inhibitor competitive with respect to the alcohol substrate. In the presence of O2, apparent Km values of 145 mM were recorded for methanol. 6. Cyanide and alphaalpha'-bipyridine were inhibitors competitive with respect to the amine activator. 7. The properties of the enzyme from Rhodopseudomonas acidophila are compared with those of similar enzymes from other organisms, and implications of the salient differences are discussed.     

Determinants in Microbial Colonization of the Murine Gastrointestinal Tract: pH, Temperature, and Energy-Yielding Metabolism of Torulopsis pintolopesii

Torulopsis pintolopesii is an indigenous yeast that colonizes the secreting epithelia in the stomachs of mice and rats. A wild-type strain of this microbe was isolated and identified. To attempt to learn characteristics of the yeast that are advantageous to it in colonizing its natural habitat in vivo, we examined some aspects of its nutrition and energy-yielding metabolism and some environmental conditions that influence its growth in vitro. The yeast appeared to be limited in the compounds it can utilize as carbon and nitrogen sources. It grew best at 37°C and did not grow at 23 or 43°C. It grew optimally at neutral pH but could grow aerobically at pH values as low as 2.0 and anaerobically at pH values as low as 3.4. As assessed by measurements of growth rates and yield coefficients, it grew better aerobically than anaerobically. When grown aerobically, it had a cyanide-sensitive system for taking up O₂ and tested positively for cytochrome c oxidase activity. A petite mutant strain isolated from the wild-type strain had a growth rate and yield coefficient when incubated aerobically that were essentially the same as those of the wild-type parent grown anaerobically. Likewise similar to the wild-type parent grown anaerobically, the petite strain, though incubated aerobically, did not take up O₂. Yeast-free mice associated with either the wild-type or the petite mutant strain were colonized at essentially the same rates and to similar final population levels by both strains. The yeast's capacity to respire may be of little advantage to it in its natural environment. By contrast, its abilities to grow best at 37°C and to grow at low pH values are undoubtedly advantageous characteristics in this respect. The limitations in its carbon and nitrogen nutrition are difficult to evaluate as ecological factors in its colonization of the natural habitat.     

Production of L-serine by the methanol utilizing bacterium,Pseudomonas 3ab

Summary A bacterium capable of growth on methanol and some organic acids as sole source of carbon and energy has been isolated and designated Pseudomonas 3ab. This facultative methylotrophic organism apparently utilizes the serine pathway of formaldehyde fixation.When methanol was used as the sole carbon source for growth, L-serine production by Pseudomonas 3ab occurred upon the addition of glycine and methanol at the end of the exponential growth phase. The maximum yield of L-serine (4.7 g/l) was obtained when 20 g/l glycine and 8 g/l methanol were added and the pH of the culture medium was changed to 8.5.Although Pseudomonas 3ab is unable to grow on L-serine or glycine, it is very active in decomposing these amino acids. The degradation of L-serine and glycine has been shown to be pH-dependent with a minimum at pH 8.5–9.0.     

Structural aspects of the dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila.

1. A dye-linked alcohol dehydrogenase was purified 60-fold from extracts of Rhodopseudomonas acidophila 10050 grown aerobically on ethanol. 2. The properties of this enzyme were identical with those of the alcohol dehydrogenase synthesized by this organism during growth on methanol anaerobically in the light, and they are judged to be the same enzyme. 3. The enzyme gave a single protein band, coincident with alcohol dehydrogenase activity, during electrophoresis on polyacrylamide gel. 4. The amino acid composition, ioselectric point, u.v. and visible absorption spectra of the enzyme were determined and compared with those of other similar enzymes. 5. The presence of 0.7--1.0 g-atom of non-haem, acidlabile iron/mol of enzyme was shown by atomic absorption spectrophotometry and colorimetric assay. The iron could not be dissociated from the enzyme by dialysis against chelating agents. 6. E.p.r. spectroscopy of the enzyme did not indicate any redox function for the iron during alcohol dehydrogenation, but showed a signal at g = 2.00 consistent with the presence of a protein-bound organic free radical. 8. Antisera were raised against alcohol (methanol) dehydrogenases purified from Rhodopseudomonas acidophila, Paracoccus denitrificans and Methylophilus methylotrophus. 9. The antiserum to the Rhodopseudomonas acidophila enzyme cross-reacted with neither of the two other antisera, nor with crude extracts of methanol-grown Hyphomicrobium X and Pseudomonas AM1, thus emphasizing its singular biochemical properties.     

Isolation and growth rates of methanol utilizing rhodospirillaceae

38 pure culture strains belonging to seven species of the Rhodospirillaceae were isolated from 39 methanol enrichment cultures inoculated with water and mud samples of different habitats. None of the strains exhibited doubling times shorter than 10 h in methanol-bicarbonate media.     

产生辅酶Q10的光合细菌菌株的分离及鉴定

对水塘污泥中富集分离的13株光合细菌产生的CoQ10进行定性定量分析,筛出CoQ10含量较高的菌株2c并对其进行系统鉴定。菌株2c为革兰氏阴性菌,细胞杆状,菌体大小0.6μm～0.9μm×1.2μm～2.0μm,单极生鞭毛,片层状光合内膜,位于细胞质膜下并与之平行。光照厌氧或黑暗好氧条件下均可生长,光照下菌体产生红色素,菌体含有细菌叶绿素a和类胡萝卜素。最适生长温度30～35℃,pH7.0～8.0。多种有机化合物均可作为光合作用的电子供体和碳源,蛋白胨和硫酸铵是其生长的较好氮源,酵母膏对其生长有明显刺激作用。16S rDNA序列系统发育分析表明,菌株2c在系统进化树上与GenBank中序列号为AY751758、DQ001155、DQ001158的沼泽红假单胞菌聚为一族。菌株2c至少能稳定传代15次。初步确定菌株2c为沼泽红假单胞菌。