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SUMMARY: Some 300 strains of bacteria representing twenty genera were grown on an acid-hydrolysed casein medium. Using the paper chromatographic technique of Woiwod (1949) a preliminary survey was made of the amino-acid and polypeptide composition of the bacterial culture filtrates. It was proved that: (a) changes may occur in the filtrate chromatogram which have group or species significance; (b) bacteria with simple nutrient requirements, i.e. which utilize ammonia, do not affect the chromatogram in the initial growth stage; (c) whenever the chromatogram is affected serine is the first amino-acid to be metabolized; (d) Gram-positive bacteria eliminate the aspartic acid spot but leave the basic amino-acid group unaffected, and the reverse occurs with Gram-negative bacteria; (e) many bacteria synthesize ninhydrin-positive material, presumably polypeptide, the synthesis of a given kind of polypep-tide being sometimes associated with a particular group or species of bacteria. By a disintegrator technique it was demonstrated that bacteria store many free amino-acids inside the cell.

Estimates were obtained of the extent to which cysteine and methionine were incorporated into the protein of the microbes of rumen digesta without prior degradation and resynthesis. By using the amino acids labeled with both 35 S and 14 C, it was observed that a large proportion of the 35 S appeared in the sulfide pool and of the 14 C appeared in volatile fatty acids. By isolating the appropriate amino acid, obtaining the 14 C to 35 S ratio, and comparing this with the ratio in the added amino acid, the degree of direct incorporation was calculated. For cysteine it was estimated that at most 1% and for methionine, at most 11% of the amino acid in the free pool was incorporated unchanged into microbial protein. As a consequence of these findings, it is considered that the method for measuring microbial protein synthesis in rumen digesta based upon incorporation of 35 S from the free sulfide pool is not seriously affected by direct utilization of sulfur amino acids arising from dietary sources.

Two pathways are involved in the fermentation of glutamate to acetate, butyrate, carbon dioxide, and ammonia—the methylaspartate and the hydroxyglutarate pathways which are used by Clostridium tetanomorphum and Peptococcus aerogenes , respectively. Although these pathways give rise to the same products, they are easily distinguished by different labeling patterns of the butyrate when [4- 14 C]glutamate is used as substrate. Schmidt degradation of the radioactive butyrate from C. tetanomorphum yielded equally labeled propionate and carbon dioxide, whereas nearly all the radioactivity of the butyrate from P. aerogenes was recovered in the corresponding propionate. This procedure was used as a test for the pathway of glutamate fermentation by 15 strains (9 species) of anaerobic bacteria. The labeling patterns of the butyrate indicate that glutamate is fermented via the methylaspartate pathway by C. tetani, C. cochlearium , and C. saccarobutyricum , and via the hydroxyglutarate pathway by Acidaminococcus fermentans, C. microsporum, Fusobacterium nucleatum , and F. fusiformis . Enzymes specific for each pathway were assayed in crude extracts of the above organisms. 3-Methylaspartase was found only in clostridia which use the methylaspartate pathway, including Clostridium SB4 and C. sticklandii , which probably degrade glutamate to acetate and carbon dioxide by using a second amino acid as hydrogen acceptor. High levels of 2-hydroxyglutarate dehydrogenase were found exclusively in organisms that use the hydroxyglutarate pathway. The data indicate that only two pathways are involved in the fermentation of glutamate by the bacteria analyzed. The methylaspartate pathway appears to be used only by species of Clostridium , whereas the hydroxyglutarate pathway is used by representatives of several genera.

A Bacillus species originally elected for the ability to utilise unbranched-alkyl-side-chain-alkylbenzene-sulphonate (ABS) isomers as the sole source of carbon and sulphur was found to be able to utilise various α-methyl-branched-alkyl-side-chain(ABS) isomers in a similar minimal nutrient role. The enzymic mechanism involved in α-methyl-branched-alkyl-side-chain biodegradation of various ABS isomers by the Bacillus was demonstrated to involve the classical β-oxidation sequence characteristic of unbranched-fatty-acid oxidation, by appropriate enzyme induction experiments. Results obtained from such enzyme induction studies plus an examination of the behaviour of these induced enzymes during separation by gel-filtration indicated a single set of enzymes to be responsible for the β-oxidation of long-chain fatty acid isomers, unbranched-alkyl-side-chain (ABS) isomers and α-methyl-branched-alkyl-side-chain (ABS) isomers in the Bacillus species. The substrate-specificity of partially purified enzymes after growth on appropriate substrates confirmed the operation in this microorganism of a single β-oxidation pathway capable of catalysing the oxidation of a wide range of different chemicals containing either unbranched or α-methyl-branched alkyl side chains.

Transfer RNA from extreme thermophilic bacteria is useful material for studies on the mechanism of protein biosynthesis at high temperature and the structurefunction relation of tRNA. There have been some reports on these problems [l-6] , but the exact nature of the RNA of thermophiles is not fully understood. In this work we used tRNA from an extreme thermo, philic bacterium Thermus thermophilus HB 8, isolated from a hot spring. This bacterium was formerly named. Flavobacterium thermophilum [7-g], and it can even grow at 85°C. The tRNA was found to contain 5-methyl2-thiouridine (m’ s2 U) in the sequence G-m5 s2 U-G-C-G. These findings strongly suggest that most of the ribothymidine normally present in the GT$C region is replaced by m5 szU in tRNA of this thermophile. The m5 s2 IJ is probably important for the capacity of the tRNA to synthesize protein at high temperature.

Butyrivibrio sp. C3 degrades rutin anaerobically to yield phloroglucinol, carbon dioxide, 3,4-dihydroxy-benzaldehyde, and 3,4-dihydroxyphenylacetic acid. The sugars are first released by intracellular glycosidases and then the heterocyclic ring of the aglycone undergoes hydrolytic cleavage. The glycosides of quercetin added to the medium are readily metabolized, but the aglycone is not.

An organism belonging toPseudomonadaceaeand capable of utilizing 3-hydroxy-3-methylglutarate as sole carbon source has been isolated from soil. Whole-cell preparations catalyze the oxidation of acetoacetate, acetate, glyoxylate, and citric acid cycle intermediates. Cell-free extracts of 3-hydroxy-3-methylglutarate-grown cells show an adenosine triphosphate, coenzyme A (CoA), and Mg2+-dependent conversion of 3-hydroxy-3-methylglutarate to 3-hydroxy-3-methylglutaryl-CoA. Succinyl-CoA-generating system has no effect on the activation and catabolism of 3-hydroxy-3-methylglutarate.

The design and construction of an anaerobic CO 2 cabinet are described. Air is displaced by a stream of oxygen-free CO 2 , and anerobic conditions are produced in 3 hr. The equipment is simple and cheap to operate and has been found to be satisfactory for the isolation of strict anaerobes from the mouse intestine.

Pseudomonas AM1 grows on β-hydroxybutyrate and methanol at similar rates. β-Hydroxybutyrate is not metabolized by way of the glyoxylate bypass, but is assimilated by the novel route (with acetate as an intermediate) that operates during growth of this organism on ethanol. Evidence from short-term labelling experiments indicates that acetate, which is a possible intermediate in the assimilation of C1 compounds, is rapidly metabolized to glycine during growth of Pseudomonas AM1 on methanol.

Soluble cell-free extracts of actinomycete S4 grown on media containing mevalonate catalyze acetoacetate formation from mevalonate, mevaldate, and β-hydroxy-β-methylglutaryl-coenzyme A (CoA). Conversion of mevalonate to acetoacetate involves formation of free β-hydroxy-β-methylglutaryl-CoA, but not free mevaldate. The reaction favors mevalonate oxidation, and nicotinamide adenine dinucleotide, rather than nicotinamide adenine dinucleotide phosphate, acts as oxidant.

Rumen microorganisms rapidly metabolize citric acid to carbon dioxide and acetic acid. The rate of metabolism varied between 0.00008 and 0.76 μmoles per g per min, the rate becoming higher as the citric acid concentration increased. The addition of potassium chloride to rumen contents decreased the rate of utilization. The results indicate that dietary citric acid is unlikely to accumulate in the rumen to a sufficiently high level to be an important factor in hypomagnesemia, except where other factors such as very high potassium levels in the food influence its metabolism.

Growing cultures, washed cells, and cell-free preparations of Gluconobacter melanogecnus IFO 3293 converted L-sorbose to 2-keto-L-gulonic acid, to D-sorbitol (which was metabolized further) and to 5-keto-D-fructose.

WE wish to emphasize the usefulness of the method of paper chromatography of sugar1,2, especially when applied on a quantitative basis3, in the study of bacterial metabolism. As an example we quote the following experiment on the utilization of sucrose by Leuconostoc mesenteroides (Lister Institute strain No. 3351).

Summary1) As in the case of animal lipids and unsaturated fatty acids, various bacterial and vegetable lipids are capable of forming yellow pigment from p-aminobenzoic acid and p-aminosalicylic acid. 2) Oxidation considerably increases the activity of certain lipids to form the pigment. This enhanced activity is apparently not due to an increase in the peroxide content but rather to the formation of other oxidation products such as fatty aldehydes or ketones. 3) These results suggest that bacterial lipids play a role in the PABA pigment formation by growing cultures of mycobacteria. 4) The possible role of lipids in bacterial metabolism as important oxido-redox systems has been discussed. 5) A new filter paper method has been applied for the study of biologic oxidations of lipids and fatty acids.

Microbes in ruminal contents incorporated 14 C into cells when they were incubated in vitro in the presence of [ 14 C]carboxyl-labeled indole-3-acetic acid (IAA). Most of the cellular 14 C was found to be in tryptophan from the protein fractions of the cells. Pure cultures of several important ruminal species did not incorporate labeled IAA, but all four strains of Ruminococcus albus tested utilized IAA for tryptophan synthesis. R. albus did not incorporate 14 C into tryptophan during growth in medium containing either labeled serine or labeled shikimic acid. The mechanism of tryptophan biosynthesis from IAA is not known but appears to be different from any described biosynthetic pathway. We propose that a reductive carboxylation, perhaps involving a low-potential electron donor such as ferredoxin, is involved.

Pseudomonas AM1 grows on ethanol with a mean generation time of about 10h. A single alcohol dehydrogenase is responsible for oxidation of both methanol and ethanol. It is proposed that the glyoxylate bypass does not operate in Pseudomonas AM1 during growth on ethanol. Although malate synthase is present in extracts of ethanol-grown Pseudomonas AM1, the activity of isocitrate lyase is negligible. Short-term incubation experiments with [14C]ethanol and [14C]acetate indicate that a novel pathway operates during growth of Pseudomonas AM1 on ethanol. Glycollate, glyoxylate and malate are probably intermediates in this pathway.

A procedure for detecting the degradation of amino acids by microorganisms is described, and examples of its use in the characterization of bacteria are presented. The procedure involves inoculating a buffered solution of amino acids with a suspension of bacteria, incubating, chromatographing a sample of the suspension, and detecting degradation in terms of absence of ninhydrin-positive spots.

An extracellular complex was isolated from the culture filtrate of Ferrobacillus ferrooxidans grown on either iron or sulfur. The complex isolated from the culture filtrate of iron-grown cells was red with an absorption maxima at 500–510 mμ. This complex consisted of approximately 60% non-phosphorus lipid. Ferrous iron was exclusively associated with a lipopolysaccharide–phospholipid fraction of the complex. The sulfur complex unlike the iron complex contained approximately 70% phospholipid and was yellowish. On the basis of hexosamine and heptose values, less lipopolysaccharide appeared to be associated with the phospholipid fraction of the complex from sulfur-grown cells than from the iron-grown cells.

The importance of the gastrointestinal microflora in the metabolism of nicotinic acid and nicotinamide has been investigated using 7-14C-labeled vitamin forms. The CO2 released from C-7 of the vitamins forms has been shown to result from microbial metabolism. The distribution of label in urinary metabolites was essentially unchanged in germ-free animals, indicating that the tissues of the rat are capable of metabolizing both vitamin forms in a normal manner without microbial assistance.

Six strains of anaerobic rumen bacteria were grown in a rumen fluid medium containing different concentrations of the urease inhibitor, acetohydroxamic acid. Bacteroides succinogenes S85 failed to grow in the presence of 1 × 10−2 M acetohydroxamic acid whereas growth by Selenomonas ruminantium GA192 was not affected when the initial concentration was 5 × 10−2 M. In cultures of the remaining strains, acetohydroxamic acid extended the lag phase without changing the growth rate in the log phase. The order of decreasing overall sensitivity to acetohydroxamic acid by the strains in terms of growth was Bacteroides succinogenes S85 [Formula: see text]Bacteroides ruminicola 23 > Butyrivibrio fibrisolvens D1 ≥ Butyrivibrio sp. C3 > Megasphaera (Peptostreptococcus) elsdenii B159 > Selenomonas ruminantium GA192. Washed cell suspensions of Butyrivibrio fibrisolvens D1, Megasphaera elsdenii B159, and Selenomonas ruminantium GA192, but of none of the other strains were capable of degrading acetohydroxamic acid. In the presence of 5 × 10−3 M acetohydroxamic acid, the extent of glucose, starch, and xylan fermentation by the strains was modified. The amounts and molar proportions of volatile fatty acids produced from these substrates were also changed. In Bacteroides ruminicola 23, the presence of acetohydroxamic acid resulted in an 89% reduction in glucose fermentation and a shift in volatile fatty acid production from propionate to acetate. Butyrivibrio fibrisolvens D1 and Butyrivibrio sp. C3 fermented more glucose, but less xylan, when acetohydroxamic acid was present; with glucose as substrate, volatile fatty acid production shifted markedly from n-butyrate to propionate. The results support the conclusion drawn from previous studies with the mixed rumen microbiota, that the potential effect of the compound upon rumen microbial activities in vivo is not limited to inhibition of urease activity.

SUMMARY : Heterotrophic bacteria were studied which produced nitrite in the presence of ammonia and in the absence of nitrate. A soil extract medium was prepared which allowed good growth as well as nitrite production in the absence of nitrate. Quantitative data are recorded showing that ammonia decreases as nitrite accumulates when four different cultures are grown in the soil medium. Resting cell studies add further evidence that some heterotrophic bacteria can convert ammonia to nitrite. A defined medium was prepared containing glucose or sodium acetate as the carbon source and NH,CI as the nitrogen source. This medium supported growth and nitrite production ; however, optimum conditions for growth were not established. Neither growth nor nitrite accumulation was as great in defined media as in soil-extract media. Results from defined media and from resting-cell studies rule out the possibility of any nitrate contamination. Early in the literature reports were published by Fremlin (1903, 1914, 1929), Makrinoff (1909), Cutler (1930), Cutler & Mukerji (1931), Cutler & Crump (1933), Crump (1935), Nechaeva (1947) and others suggesting the possibility of nitrification by heterotrophic bacteria. Recently Fisher, Fisher & Appleman (1952) and Hutton & ZoBell (1953) have added more data supporting these early observations. Isenberg et al. (1954) described a streptomycete which produces nitrite from urethane. This paper presents evidence that certain Gram-negative bacteria isolated from soil form nitrite in the presence of ammonia in defined and undefined media. Ammonia disappears as nitrite is produced. Special precautions have been taken to exclude nitrate from the media. METHODS Bacteria. Sixteen nitrite-producing cultures were obtained from soil by enrichment techniques. These were shown to be pure. The organisms were found to be Gram-negative short rods probably belonging to group 2 or 3 of Taylor & Lochhead's (1938) classification of soil bacteria. This classification, based on morphology and Gram-staining reactions of cultures grown on soil extract semi-solid agar, has eight large subdivisions, group 2 consisting of short Gram-negative rods and group 3 composed of short Gram-variable rods.

SUMMARY: Paper chromatograms of hydrolysates of 118 micro-organisms were examined in a study of the distribution of α, e-diaminopimelic acid and other amino-acids. A method for the identification of α, e-diaminopimelic acid is described. Diaminopimelic acid was found in nearly all the bacteria examined, except for the Gram-positive cocci, Streptomyces spp., and Actinomyces spp. It was also found in blue-green algae but in no other algae, nor in fungi, yeasts, plant viruses, or protozoa. Each species examined showed a different amino acid composition. β-Alanine and α- and γ-aminobutyric acids were sometimes found, often in several species of the same genus. Seven unidentified ninhydrin-reacting spots were recorded; none of them had the wide distribution of diaminopimelic acid.

In pruvious papers, Miura (J.J. s. 14, 103, '65) Miura and Fujimaki (J.J. s. s. 15, 257, '66) reported that genus neisseria isolated from oral materials has cytochrome respiratory system.In the present study of same bacterial metabolism, I found to have another metabolic pathway differ from cytochrome respiratory system when glucose was added to reaction mixtures as snbstrate in routine biochemical method, and obtained the following results:1. the resting cell of genus neisseria may be well utilized glucose.2. the added glucose utilization by the resting cell manometrically was not always coinsides with O2 consumption3. when the suitable concentration of NaN3 and DNP was added to reaction mixuture, after some time, O2 consumption increased rapidly.From these results, it may be seem to have the oxidative assimilation of glucose in oral bacteria.

SUMMARY: Hydrogen sulphide production by growing cultures and non-multiplying suspensions was compared and the factors influencing the sensitivity of the tests were investigated. Cysteine hydrochloride (0.01%) was added to Lemco broth to provide a medium with a standard source of sulphur. H2S was detected with lead acetate papers more readily than by lead acetate agar. Suspensions were tested with cysteine, sodium thiosulphate and sodium sulphite; the organisms investigated were mainly Bacteriaceae and seldom failed to produce H2S from cysteine; sodium thiosulphate was less readily attacked; sodium sulphite was unsuitable for this technique. Media commonly used for testing the capacity of bacteria to produce H2S are reviewed and the value of this test in bacterial classification discussed.

There exists experimental evidence for the view that phage production involves the reaction: Inactive intracellular phage precursor+ phage→ phage.1 The phage precursor has been shown to develop whenever the environmental conditions favor active bacterial metabolism and growth.2 We term cells which contain a store of precursor “activated bacteria”, and have found that the precursor content of the bacteria is very thermolabile, being destroyed at rapid rates when the cells are heated to 45°-5O°C.3 At these temperatures destruction of the precursor takes place long before sufficient damage is done to the cells to destroy their reproductive mechanism. The critical thermal increment for heat inactivation is 90,000, suggesting that the precursor either is a protein or contains a protein.In the present paper we wish to report experiments in which destruction of precursor was brought about by concentrations of iodoacetic acid that were not lethal for the bacteria used.Activated staphylococci were prepared by grow...

Although Pasteurella pestis has been the subject of a great deal of medical and immunological research, very little work has been done on the physiology of the organism itself. During recent years, attempts have been made to apply technics used in the study of bacterial metabolism to certain pathogenic organisms. The experiments reported in this paper were designed to establish the basic growth requirements of the plague bacillus, and to investigate to some extent the nature of its dissimilatory processes.Rao1 claims that 3 amino acids, cystine, phenylalanine, and proline, are essential for the development of P. pestis, and that haematin and riboflavin may be considered as accessory growth factors. However, he does not give evidence that the organism can grow with a mixture of these compounds alone, and provides neither a carbon nor a nitrogen source other than amino acids in most of his media. In an excellent study dealing with the nutrition of the members of the genus Pasteurella, Berkman2 could find no...

SUMMARY: The rates at which two strains each of four species of cellulolytic rumen bacteria could grow on and solubilize ball-milled filter paper were compared in vitro. Strains of Ruminococcus albus solubilized it most rapidly, followed by R. flavefaciens. Butyrivibrio fibrisolvens showed consistently low rates of solubilization while strains of Cillobacterium cellulosolvens maintained on cellobiose-containing medium gradually lost their ability to solubilize cellulose. Cellulolysis could not be restored by successive transfers to media each containing one of different cellulose oligosaccharides. Strains of R. albus and R. flavefaciens grew almost as rapidly in cellulose- as in cellobiose-containing media, while B. fibrisolvens strains grew better in medium with cellobiose. On this basis the ruminococci were termed true cellulolytic bacteria.

This review covers in part the literature appearing during 1951 , and is restricted primarily although not entirely to bacterial metabolism. As in previous years, the field of metabolism moved too rapidly for total coverage to be achieved in the space limitations imposed. Certain areas of metabolism are treated more extensively than others because of their importance, and attempts are made when possible to relate papers and to comment on their significance.