Conversion of choline methyl groups through trimethylamine into methane in the rumen.

1. Choline methyl groups were rapidly metabolized to trimethylamine by rumen micro-organisms. 2. Trimethylamine was further metabolized to methane, but this system was more easily saturated by an excess of substrate, so that trimethylamine accumulated in the rumen of the fed animal. 3. Although trimethylamine was the only intermediate isolated in the conversion of the methyl groups of choline into methane, methylamine also served as a substrate for methane production. 4. The methyl group of methionine was also converted into methane by rumen fluid, but the methyl groups of carnitine were not.     

Salivary film expresses a complex, macromolecular binding site for Streptococcus sanguis

Teeth in the oral cavity are coated with a salivary film or pellicle, which lacks apparent intermolecular organization. This heterogeneous film facilitates binding of early commensal colonizing bacteria, including Streptococcus sanguis. To test the hypothesis that sufficient intermolecular organization exists in salivary films to form binding sites for S. sanguis, an in vitro model of saliva-coated teeth was probed with murine anti-idiotypical monoclonal antibodies (mAb2, anti-ids). The anti-ids were harvested from hybridomas that were developed in response to first generation murine hybridomas that produced anti-S. sanguis adhesin monoclonal antibodies (mAb1). The anti-ids (i) reacted with experimental salivary films and inhibited S. sanguis adhesion in a dose-dependent fashion. In Western blots, the anti-ids (ii) recognized a high molecular weight salivary antigen and (iii) secretory IgA (sIgA) light chain and alpha-amylase. After isolation by gel filtration from whole saliva or mixed secretory IgA and alpha-amylase, the high molecular weight component, containing amylase activity and sIgA, bound to hydroxyapatite to promote adhesion of S. sanguis. Therefore, a complex enriched in secretory immunoglobulin A and alpha-amylase forms a S. sanguis-binding site.     

Fimbria-associated proteins of Bacteroides loescheii PK1295 mediate intergeneric coaggregations

Bacteroides loescheii PK1295 serves as a coaggregation bridge between Streptococcus sanguis 34 and Actinomyces israelii PK14, two gram-positive oral bacteria that are otherwise unable to coaggregate. Whereas coaggregation with S. sanguis 34 is inhibited by lactose, no simple sugar was found that inhibited coaggregation with A. israelii PK14. Coaggregation-defective (Cog-) mutants of B. loescheii PK1295 were isolated for the purpose of identifying the surface components responsible for the interaction with each coaggregation partner. Selection for spontaneously occurring Cog- mutants gave rise to two phenotypic classes of mutants. Type I lost the ability to coaggregate with S. sanguis 34, whereas type II failed to coaggregate with either S. sanguis 34 or A. israelii PK14. Purified fimbriae from the parent agglutinated cells of both partners, and agglutination with S. sanguis 34 was inhibited by lactose. Denaturing polyacrylamide gel electrophoresis and immunoblot analysis demonstrated the presence of both a 75- and a 43-kilodalton (kDa) protein associated with parental fimbriae, but only a 43-kDa protein was seen with fimbriae prepared from the type I mutant. Neither polypeptide was found in similar preparations from the type II mutants. Our data suggest that coaggregation of B. loescheii PK1295 with both gram-positive partners is mediated by fimbria-associated proteins present on the surface of the gram-negative organism and that the 75- and 43-kDa polypeptides are responsible for the recognition of S. sanguis 34 and A. israelii PK14 cells, respectively.     

Transformation of Streptococcus sanguis Challis by plasmid deoxyribonucleic acid from Streptococcus faecalis.

Plasmid deoxyribonucleic acid (DNA) from Streptococcus faecalis, strain DS5, was transferred to the Challis strain of Streptococcus sanguis by transformation. Two antibiotic resistance markers carried by the beta plasmid from strain DS5, erythromycin and lincomycin, were transferred to S. sanguis at a maximum frequency of 1.8 x 10-5/colony-forming unit. Approximately 70% of the covalently closed circular DNA isolated from transformant cultures by dye buoyant density gradients was shown to be hybridizable to beta plasmid DNA. Two major differences were observed between the beta plasmid from S. faecalis and the plasmid isolated from transformed S. sanguis: (i) the beta plasmid from strain DS5 sedimented in velocity gradients at 43S, whereas the covalently closed circular DNA from transformed Challis sedimented at 41S, suggesting a 1.5-Mdal deletion from the beta plasmid occurred; (ii) although the 43S beta plasmid remained in the supercoiled configuration for several weeks after isolation, the 41S plasmid was rapidly converted to a linear double-stranded molecule. Attempts to transform S. sanguis with the alpha plasmid from S. faecalis, strain DS5, were unsuccessful.     

Methanogenesis from Choline by a Coculture of Desulfovibrio sp. and Methanosarcina barkeri

A sulfate-reducing vibrio was isolated from a methanogenic enrichment with choline as the sole added organic substrate. This organism was identified as a member of the genus Desulfovibrio and was designated Desulfovibrio strain G1. In a defined medium devoid of sulfate, a pure culture of Desulfovibrio strain G1 fermented choline to trimethylamine, acetate, and ethanol. In the presence of sulfate, more acetate and less ethanol were formed from choline than in the absence of sulfate. When grown in a medium containing sulfate, a coculture of Desulfovibrio strain G1 and Methanosarcina barkeri strain Fusaro degraded choline almost completely to methane, ammonia, and hydrogen sulfide and presumably to carbon dioxide. Methanogenesis occurred in two distinct phases separated by a lag of about 6 days. During the first phase of methanogenesis choline was completely converted to trimethylamine, acetate, hydrogen sulfide, and traces of ethanol by the desulfovibrio. M. barkeri fermented trimethylamine to methane, ammonia, and presumably carbon dioxide via dimethyl- and methylamine as intermediates. Simultaneously, about 60% of the acetate expected was metabolized. In the second phase of methanogenesis, the residual acetate was almost completely catabolized.     

Cross-reactive immunodeterminants on Streptococcus sanguis and collagen. Predicting a structural motif of platelet-interactive domains.

Cross-reactive immunodeterminants on a fibril-associated surface antigen of Streptococcus sanguis and types I and III collagen participate in the induction of aggregation of human platelets. To further understand the basis for this apparent molecular mimicry, antitype-specific collagen antibodies, anti-KPGEPGPK (an analogue of platelet-interactive domains on collagen) and a panel of KPGEPGPK-like synthetic peptides were used as probes. When collagen or S. sanguis cells were pretreated with the anti-collagen antisera, the induction of aggregation of platelet-rich plasma was greatly delayed or abrogated. These anti-collagen antibodies also neutralized KPGEPGPK and purified S. sanguis platelet-interactive antigens as inhibitors of S. sanguis or collagen-induced aggregation of platelets in plasma. In immunoblot analyses, these anti-collagen antibodies reacted with S. sanguis platelet-interactive antigens. Additionally, antisera against the platelet-interactive antigen of S. sanguis selectively reacted with undigested type I collagen and with fragments CB3 and CB6 of cyanogen bromide-treated type I collagen. Finally, when platelets were pretreated with synthetic peptides containing specific amino acid substitutions within the KPGEPGPK sequence, the time to onset of platelet-rich plasma aggregation by both agonists was altered. The hierarchical pattern of responses of platelets to these peptides and predictions of the structural changes produced by simulated insertions of each peptide into the CB4 sequence of type III collagen suggested conformational requirements for interactions with platelets. Thus, these data show that cross-reactive immunodeterminants of S. sanguis and collagen induce platelet aggregation. The platelet-interactive domains are predicted to be characterized by a structural motif with the consensus sequence X-P-G-E-P/Q-G-P-X.     

The effect of lowering the pH on the composition and metabolism of a community of nine oral bacteria grown in a chemostat

Nine oral bacteria, associated with both healthy and diseased sites in the mouth, were grown at D = 0.05 h-1 (mean generation time 13.9 h) in a glucose-limited chemostat. After an initial period of steady-state growth at pH 7.0, pH control was discontinued. The pH then decreased until it stabilized at pH 4.1 after 9 d (16 generations), while the Eh rose from -165 mV to +160 mV. The lowering in pH resulted in the composition and metabolism of the flora being altered and in increased bacterial aggregation. At pH 7.0, 'Streptococcus mitior', Veillonella alcalescens and S. sanguis were most numerous while at pH 4.1 the counts of all bacteria fell except for Lactobacillus casei, which became predominant. The proportions of S. mutans within the community also increased while S. sanguis was recovered only occasionally and Bacteroides intermedius was not detected below pH 4.6. The survival at pH 4.1 of several other species would not have been predicted from earlier pure culture studies. Relative to pH 7.0, the community growing at pH 4.1 produced more lactic acid, washed cells had a greater glycolytic activity over a wider pH range but amino acid metabolism decreased. In general, when pH control was restored, so were the original patterns of metabolism and bacterial counts, except for B. intermedius, which was still not detected. The inverse relationship between S. sanguis and S. mutans, and the increase in proportions of L. casei and S. mutans during growth in a low pH environment parallel observations made in vivo and suggest that the chemostat can be used as a model for microbial behaviour in dental plaque.     

Efficient synthesis of the cholinephosphate phospholipid headgroup

In search of an efficient method to prepare cholinephosphate headgroups in phospholipids under mild conditions (where the diacylglycerol moiety is not subject to oxidation), a method was developed for phosphorylation using a trialkyl phosphite and I2. The active intermediate is a phosphoryl iodide formed by oxidation of the phosphite with I2. 2-Bromoethanol, dimethyl chlorophosphite, and an alcohol (diglyceride) are converted to a phosphate triester in a one-pot reaction with high yield. In the second reaction, the phosphate triester is demethylated, and the ethyl bromide group is converted to choline by treatment with aqueous trimethylamine. This procedure is applied to the synthesis of hexadecylphosphocholine, and 1,2-didecanoyl-1-deoxy-1-thio-sn-glyceryo-3-phosphocholine.     

健康青少年口腔优势菌的分离、培养和鉴定

目的 寻找健康青少年口腔优势菌,为口腔微生态调节剂的研究提供基础资料。方法 通过需氧及厌氧培养法,随意引物聚合酶链反应法来得到健康青少年口腔的优势菌。结果 实验发现,在健康青少年口腔中可以分离出1种构成比较大的细菌,经鉴定为1种血链球菌。结论 血链球菌是健康青少年口腔优势菌。     

Transport characteristics of system A in the rat exocrine pancreatic epithelium analyzed using the specific non-metabolized amino acid analogue alpha-methylaminoisobutyric acid

The selectivity and kinetics of system A amino acid transport in the rat exocrine pancreatic epithelium were characterized using the specific analogue alpha-methylaminoisobutyric acid. Unidirectional influx of alpha-methylaminoisobutyric acid was measured in isolated perfused pancreata by rapid dual tracer dilution. In cross-inhibition experiments DL-methylalanine, L-serine, L-cysteine, glycine, L-phenylalanine and L-glutamine were effective inhibitors of influx, whereas L-glutamate and L-lysine were less effective. In the presence of sodium alpha-methylaminoisobutyric acid influx was saturable with an apparent Kt = 1.7 +/- 0.2 mM and Vmax = 0.49 +/- 0.03 mumol/min per g (mean +/- S.E., n = 6). Influx of alpha-methylaminoisobutyric acid at 50 microM and 100 microM concentrations was significantly inhibited as the perfusate sodium concentration was gradually decreased from 156 mM to 26 mM by isoosmolar choline replacement. Estimated Kt values for sodium at these two methylaminoisobutyric acid concentrations approximated 200 mM. System A activity in the basolateral membrane of the exocrine pancreatic epithelium exhibits a high transport affinity, a wide tolerance for different amino acids and a dependency upon the extracellular sodium concentration.     

Relatedness between Streptococcus pneumoniae and viridans streptococci: transfer of penicillin resistance determinants and immunological similarities of penicillin-binding proteins

The occurrence of highly variable penicillin-binding proteins (PBPs) in penicillin-resistant Streptococcus pneumoniae suggested that transfer of homologous genes from related species may be involved in resistance development. Antiserum and monoclonal antibodies raised against PBPs 1a and 2b from the susceptible S. pneumoniae R6 strain were used to identify related PBPs in 41 S. mitis, S. sanguis I and S. sanguis II strains mostly isolated in South Africa with MIC values ranging from less than 0.15 to 16 mg/ml. Furthermore, the possibility of genetic exchange was examined with 30 penicillin-resistant strains of this collection (MIC greater than 0.06 mg/ml) as donors using S. pneumoniae R6 as recipient in transformation experiments. The majority of S. mitis and S. sanguis II strains but none of the S. sanguis I strains could transform penicillin resistance genes into S. pneumoniae R6. All positive donor strains and all susceptible isolates of S. mitis and S. sanguis II strains contained PBPs which cross-reacted with the anti-PBP 1a and/or anti-PBP 2b antibodies. On the other hand, only five of the 14 S. sanguis I strains contained a PBP that reacted with one of the antibodies. This strongly suggested the presence of genes homologous to the pneumococcal PBP 1a and 2b genes in viridans streptococci, and documents that penicillin resistance determinants can be transformed from viridans streptococci into the pneumococcus.     

Deuterium isotope effects during carbon-hydrogen bond cleavage by trimethylamine dehydrogenase. Implications for mechanism and vibrationally assisted hydrogen tunneling in wild-type and mutant enzymes

His-172 and Tyr-169 are components of a triad in the active site of trimethylamine dehydrogenase (TMADH) comprising Asp-267, His-172, and Tyr-169. Stopped-flow kinetic studies with trimethylamine as substrate have indicated that mutation of His-172 to Gln reduces the limiting rate constant for flavin reduction approximately 10-fold (Basran, J., Sutcliffe, M. J., Hille, R., and Scrutton, N. S. (1999) Biochem. J. 341, 307-314). A kinetic isotope effect (KIE = k(H)/k(D)) accompanies flavin reduction by H172Q TMADH, the magnitude of which varies significantly with solution pH. With trimethylamine, flavin reduction by H172Q TMADH is controlled by a single macroscopic ionization (pK(a) = 6.8 +/- 0.1). This ionization is perturbed (pK(a) = 7.4 +/- 0.1) in reactions with perdeuterated trimethylamine and is responsible for the apparent variation in the KIE with solution pH. At pH 9.5, where the functional group controlling flavin reduction is fully ionized, the KIE is independent of temperature in the range 277-297 K, consistent with vibrationally assisted hydrogen tunneling during breakage of the substrate C-H bond. Y169F TMADH is approximately 4-fold more compromised than H172Q TMADH for hydrogen transfer, which occurs non-classically. Studies with Y169F TMADH suggest partial thermal excitation of substrate prior to hydrogen tunneling by a vibrationally assisted mechanism. Our studies illustrate the varied effects of compromising mutations on tunneling regimes in enzyme molecules.     

A collagen-like immunodeterminant on the surface of Streptococcus sanguis induces platelet aggregation

The basis of similarities in the mechanism of human platelet aggregation induced by soluble collagen and the dental plaque bacterium Streptococcus sanguis was analyzed. Structural and functional comparisons were made by using molecular probes, including rabbit antibody fractions reactive with components on S. sanguis and a synthetic, collagen-like octapeptide mimicking segments from cyanogen bromide fragments 6 and 4 of types I and III collagen, respectively. When platelets were pretreated with tryptic peptides or class II antigen of S. sanguis or with the synthetic, collagen-like octapeptide, the onset of aggregation in response to S. sanguis and collagen was prolonged. When compared to other peptides of similar size and charge, the collagen-like peptide's action towards platelets was shown to be selective. Indeed, absorption of antiserum to S. sanguis cells with particulate type I collagen removed specificities directed at a single S. sanguis antigen. These observations suggested that a common platelet-interactive immunodeterminant on soluble types I and III collagens, particulate type I collagen, and S. sanguis cells was present. Selective inhibition by antibody was used to show structural similarities between the S. sanguis surface proteins and collagen. When either agonist was pretreated with anti-S. sanguis IgG or Fab fragments, the lag time to onset of platelet aggregation was increased. Greater increases in the lag time to aggregation was seen when S. sanguis cells or collagen were pretreated with anti-S. sanguis IgG or Fab fragments made relatively specific for the class II antigen. Neutralization of the platelet-interactive action of the octapeptide by anti-S. sanguis antibody fractions showed that the immunodeterminant common to S. sanguis and collagen triggered platelets in plasma to aggregate. Although the anti-S. sanguis antibodies could inhibit fibrillogenesis, this action was apparently independent of interactions with platelets. In contrast, S. sanguis could bind or adhere to platelets by different determinants. Our data suggest that platelets have at least two distinct sites that bind collagen or S. sanguis. One of these may be a common site for collagen and S. sanguis agonists.     

Genetic and biochemical characterization of the F-ATPase operon from Streptococcus sanguis 10904

Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species Streptococcus sanguis and Streptococcus mutans. Differences in the pH optima of the respective ATPases appears to be the main reason that S. mutans is more tolerant of low pH values than S. sanguis and hence pathogenic. We have recently reported the genetic arrangement for the S. mutans operon. For purposes of comparative structural biology we have also investigated the F-ATPase from S. sanguis. Here, we report the genetic characterization and expression in Escherichia coli of the S. sanguis ATPase operon. Sequence analysis showed a gene order of atpEBFHAGDC and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both S. sanguis and S. mutans; however, it is not induced to the same extent in the nonpathogenic S. sanguis. Expression studies with an atpD deletion strain of E. coli showed that S. sanguis-E. coli hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits.     

人肠道梭菌和甲烷马赛球菌协同代谢甜菜碱和胆碱产甲烷研究

【目的】根据人肠道富含胆碱和甜菜碱,同时肠道微生物组中具有裂解胆碱和还原甜菜碱产三甲胺的细菌,以及利用三甲胺产甲烷的古菌,本研究探讨肠道细菌与古菌协同代谢甜菜碱和胆碱产甲烷的可能性。【方法】调查不同年龄段人群粪便中的16S rRNA基因多样性,分析肠道中古菌的菌群组成;利用定量PCR(quantitativePCR,qPCR)定量甲烷马赛球菌(Methanomassiliicoccus)特异的甲醇甲基转移酶基因mtaB和甲烷八叠球菌(Methanosarcina)及细菌的16SrRNA基因拷贝数,分析肠道中甲基营养型产甲烷古菌及总细菌的含量;宏基因组组装基因组(metagenome-assembled genomes, MAGs)分析携带甜菜碱还原酶基因grdH和胆碱裂解酶基因cutC的细菌组成。从粪便中分离代谢甜菜碱及胆碱产生三甲胺的细菌,并与分离自人肠道的甲烷马赛球菌构建共培养物,测定其协同转化甜菜碱和胆碱产甲烷的能力。【结果】年轻人粪便中含有甲烷杆菌科(Methanobacteriaceae,82.16%)的甲烷短杆菌属(Methanobrevibacter,49.18%)和甲烷...     

Kinetics of Choline Uptake into Isolated Rat Forebrain Microvessels: Evidence of Endocrine Modulation

The active uptake of [methyl-3H]choline into isolated rat brain microvessel suspension was studied as a likely guide to the transport of choline across the blood-brain barrier. The method consisted primarily of incubation of the suspension with a fixed concentration of labeled choline in the presence of increasing concentrations of unlabeled choline or any other inhibitor (I) of active uptake, defined as the difference in uptake at 37 degrees and 0 degrees C. From the linear regression of (1/V) against [I], the following values of Vmax (nmol g-1 min-1) and Km (microM) were obtained for choline: 2-month-old males, 10.6 +/- 3.8 and 6.1 +/- 0.9; 3-month old random females, 28.4 +/- 5.9 and 12.6 +/- 4.0; females at metaestrus, 17.8 +/- 10.3 and 8.3 +/- 5.0; at diestrus, 31.1 +/- 9.3 and 13.0 +/- 2.6; at proestrus, 54.9 +/- 2.2 and 14.0 +/- 1.5; at estrus, 19.2 +/- 2.2 and 2.6 +/- 1.7. The differences between males and random females (p less than 0.018) and between females at proestrus and estrus (p less than 0.005) are significant. It is suggested that these inter- and intrasex variations in choline uptake reflect a dynamic adjustment of supply in accordance with brain demand for choline at the time of assay. Hemicholinium-3 was an effective inhibitor of choline uptake, Ki = 14.0 +/- 8.5 microM; dimethylaminoethanol was much less effective; and imipramine had no measurable effect.     

Efficiency of Various Growth Media in Recovering Oral Bacterial Flora from Human Dental Plaque

MM10 sucrose blood agar (MM10 SB agar), N(2)C agar, Schaedler agar (SH agar), and mitis salivarius agar (MS agar) were tested for their ability to recover human dental plaque flora by a continuous anaerobic procedure and by a conventional anaerobic method. MM10 SB agar yielded higher recovery of bacteria from plaque samples as determined by the enumeration of colony-forming units (CFU). The CFU on N(2)C agar, SH agar, and MS agar were lower than MM10 SB agar when the continuous anaerobic procedure was used. The superior performance of MM10 SB agar was much more apparent when used for the cultivation of dental plaque by the conventional anaerobic method. Under these conditions the counts were consistently higher on MM10 SB agar as compared to the other media tested. However, the differential counts of Streptococcus sanguis and S. mutans from carious plaque samples were in general comparable on all culture media. Deletion of blood from MM10 SB agar did not lower counts. The elimination of dithiothreitol from this medium resulted in a significantly lower recovery of bacteria from the plaque samples when cultured by the conventional anaerobic method. The storage of MM10 SB agar for varying periods of time aerobic conditions did not seem to affect its performance. These findings suggest that MM10 SB agar is an ideal culture medium for the isolation, nonselective enumeration, and differential counts of bacteria present in normal and disease-associated plaques.     

Mechanism of biosynthesis of trimethylamine oxide from choline in the teleost tilapia, Oreochromis niloticus, under freshwater conditions

The mechanism of biosynthesis of trimethylamine oxide (TMAO) from dietary precursors in the teleost tilapia (Oreochromis niloticus) was investigated. Diets supplemented with quaternary ammonium choline, glycine betaine, carnitine or phosphatidylcholine were administered and significant increases in TMAO levels in the muscle were only observed with choline. [Methyl-14C] and [1,2-14C] cholines were given through dietary and intraperitoneal injection routes, but 14C-TMAO was detected only in fish with dietary administration of [methyl-14C] choline. Dietary treatment with [15N] choline resulted in the formation of [15N] TMAO in the muscle. The incorporation of radioactivity into TMAO was also observed both following dietary administration and intraperitoneal injection of [14C] trimethylamine (TMA). When choline was introduced into the isolated intestine, marked increases in TMA levels occurred. These increases were significantly suppressed in the presence of penicillin. [14C]-TMA derived from [methyl-14C] choline was detected in the cavity of the isolated intestine. The introduction of [15N] choline into the intestinal cavity resulted in the formation of [15N] TMA. TMA mono-oxygenase activities were detected in the liver and kidney. We conclude that tilapia possess the ability to produce TMAO from choline, which is related to intestinal microorganisms and tissue mono-oxygenase under freshwater conditions.     

Formation of ethylene glycol and trimethylamine from choline by Candida tropicalis

Abstract The degradation of choline by Candida tropicalis cells grown in a medium containing choline as a nitrogen source was examined. The degradation of choline by resting cells was stimulated by the addition of Cu²⁺ or glutathione, and inhibited by 2-mercaptoethanol or potassium cyanide. With feeding of [1,2-¹⁴C]choline in the resting cell reaction, the release of ¹⁴C-labelled ethylene glycol was observed on radio-gas-liquid chromatography. Ethylene glycol, as one of the degradation products, was also observed on thin-layer and gas-liquid chromatographies, and mass spectrometry. Thus, it is suggested that choline is converted to ethylene glycol and trimethylamine by C. tropicalis .     

Adenosine 3′:5′-cyclic monophosphate in young and senescent human fibroblasts during growth and stationary phase in vitro. Effects of prostaglandin E1 and of adrenaline

1. A mono-oxygenase, which oxidizes trimethylamine and other tertiary amines bearing methyl or ethyl groups, was partially purified sixfold from Pseudomonas aminovorans grown on trimethylamine as sole carbon source. 2. The preferred electron donor was NADPH. The enzyme had a pH optimum of 8.0-9.4 for trimethylamine oxidation, and 8.8-9.2 for dimethylamine oxidation. 3. The oxidation product of trimethylamine was shown to be trimethylamine N-oxide. Other tertiary amines were probably also converted into N-oxides. 4. The enzyme also oxidized secondary amines. 5. The oxidation of trimethylamine was only slightly inhibited by CO and not at all by KCN or proadifen hydrochloride (SKF 525-A), but was inhibited by trimethylsulphonium chloride, tetramethylammonium chloride, 2,4-dichloro-6-phenylphenoxyethylamine (Lilly 53325) and its NN-diethyl derivative (Lilly 18947). 6. The oxidation of dimethylamine showed a similar response to inhibitors and a parallel loss in activity on heating at 35 degrees C. 7. The activities of the trimethylamine mono-oxygenase, trimethylamine N-oxide demethylase and the secondary-amine mono-oxygenase increased severalfold during adaptation of succinate-grown bacteria to growth on trimethylamine, and the trimethylamine mono-oxygenase was the first enzyme to show an increase in activity. It is concluded that all three enzymes are involved in growth on trimethylamine by this organism.