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PubMed2005

Production of antimicrobial substances by Bacillus subtilis LFE-1, B. firmus HO-1 and B. licheniformis T6-5 isolated from an oil reservoir in Brazil.

AIMS: Forty Bacillus strains isolated from a Brazilian oil reservoir were tested against each other to select strains producing antimicrobial substances (AMS). Three strains, Bacillus subtilis (LFE-1), Bacillus firmus (H2O-1) and Bacillus licheniformis (T6-5), were selected due to their ability to inhibit more than 65% of the Bacillus strains tested. These three strains were also investigated for their capability to inhibit sulphate-reducing bacteria (SRB). Furthermore, physiological and biochemical characteristics of the antimicrobial compounds produced by the selected strains were determined. METHODS AND RESULTS: Among the forty strains tested, 36 (90%) strains were able to inhibit at least one Bacillus strain used as indicator in plate assays and three of them (LFE-1, T6-5 and H2O-1) were able to inhibit 65, 70 and 97.5% of the 40 strains studied here respectively. Clear zones of inhibition were observed when H2O-1 was tested against SRB-containing consortium T6-lab and Desulfovibrio alaskensis strain NCIMB 13491, while strain T6-5 was able to inhibit only the D. alaskensis strain. The three substances showed to be insensitive to different enzymes and chemicals, were heat stable and the substances produced by strains T6-5 and H2O-1 were active over a wide pH range. CONCLUSIONS: Three different AMS produced by Bacillus strains from an oil reservoir, two of them with activity against SRB, are presented here. SIGNIFICANCE AND IMPACT OF THE STUDY: The preliminary characterization of these AMS points to their potential use as biocides in the petroleum industry for controlling problems associated with SRB.

Bacillus↗
PubMed1979

The spectrum of Bacillus bacteremias in heroin addicts.

Bacillus bacteremias occurred in two heroin addicts. The first patient had one day of fever and chills after intravenous heroin use. Persistent cereus bacteremia consistent with endocarditis was documented and responded to four weeks of antibiotic therapy. The second patient had non-cereus Bacillus species isolated from blood cultures three times over eight days, each time after renewed heroin use. The patient remained well, and the bacteremias cleared spontaneously. Because Bacillus species frequently contaminate heroin injection materials and because the Bacillus bacteremias were temporally associated with intravenous heroin use, Bacillus bacteremias in both patients probably eventuated from heroin abuse. These cases, in conjunction with two previously reported cases of Bacillus endocarditis in heroin addicts, suggest that heroin addicts are at risk for developing Bacillus bacteremias, which may vary in severity from endocarditis to benign transient bacteremias.

Bacillus↗
PubMed1987

[Homologous and heterologous transcription of the Cry+-plasmid in Bacillus thuringiensis].

The possibility of homologous and heterologous transception of Cry+ plasmids in Bacillus thuringiensis is demonstrated. Cry+ plasmids from crystal bearing strain of Bacillus thuringiensis were transferred into acrystalline strain belonging to H5 serotype by mutual incubation. The donor strain was previously marked by the transmissive plasmid pAM beta 1 coding for erythromycin and lincomycin resistance. The transcipients having acquired the ability to synthesize delta-endotoxin were referred to H5 serotype due to their phenotype. By analogous method Cry+ plasmid was transferred from Bacillus thuringiensis to Bacillus cereus. Bacillus cereus strain GP7 was used as a recipient strain resistant to tetracycline. The presence of delta-endotoxin in transcipients was confirmed by bioprobes and immunoenzyme assay. To prove the transfer of Cry+ plasmid the plasmid profiles of the parent strains and transcipients have been analyzed. The formation of cellular contacts during mutual incubation of Bacillus thuringiensis and Bacillus cereus strains was demonstrated by electron microscopic study of ultrafine cuts.

Bacillus thuringiensis↗
PubMed1999

The hemolytic enterotoxin HBL is broadly distributed among species of the Bacillus cereus group.

The prevalence of the hemolytic enterotoxin complex HBL was determined in all species of the Bacillus cereus group with the exception of Bacillus anthracis. hblA, encoding the binding subunit B, was detected by PCR and Southern analysis and was confirmed by partial sequencing of 18 strains. The sequences formed two clusters, one including B. cereus and Bacillus thuringiensis strains and the other one consisting of Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis strains. From eight B. thuringiensis strains, the enterotoxin gene hblA could be amplified. Seven of them also expressed the complete HBL complex as determined with specific antibodies against the L(1), L(2), and B components. Eleven of 16 B. mycoides strains, all 3 B. pseudomyoides strains, 9 of 15 B. weihenstephanensis strains, and 10 of 23 B. cereus strains carried hblA. While HBL was not expressed in the B. pseudomycoides strains, the molecular assays were in accordance with the immunological assays for the majority of the remaining strains. In summary, the hemolytic enterotoxin HBL seems to be broadly distributed among strains of the B. cereus group and relates neither to a certain species nor to a specific environment. The consequences of this finding for food safety considerations need to be evaluated.

Animals↗
PubMed2000

Homoduplex and heteroduplex polymorphisms of the amplified ribosomal 16S-23S internal transcribed spacers describe genetic relationships in the "Bacillus cereus group".

Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the "B. cereus group," advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as B. anthracis or serotypes of B. thuringiensis. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The second included B. cereus and B. thuringiensis, B. anthracis appeared as a lineage of B. cereus.

Bacillus↗
PubMed2006

Enhancement of gamma-aminobutyric acid production in Chungkukjang by applying a Bacillus subtilis strain expressing glutamate decarboxylase from Lactobacillus brevis.

For a foreign glutamate decarboxylase (GAD) to be expressed in Bacillus host system, a recombinant DNA (pLip/LbGAD) was constructed by ligating an LbGAD gene from Lactobacillus brevis OPK-3 into Escherichia coli-Bacillus shuttle vector, pLip. The pLip/LbGAD construct was then transformed into Bacillus subtilis. The culture of the transformed Bacillus strain with the pLip/LbGAD construct had higher GAD activity and gamma-aminobutyric acid (GABA) concentration than those of untransformed Bacillus counterpart. In addition, Chungkukjang, a traditional Korean fermented soybean product prepared by the transformed Bacillus subtilis, contained a significantly higher level of GABA than conventional ones. Thus, by introducing a foreign GAD gene, Bacillus strains have been genetically engineered to produce high levels of GAD and GABA.

Bacillus subtilis↗
PubMed1993

[The nutritive efficiency of Bacillus cereus as a probiotic in the raising of piglets. 1. Effect on the growth parameters and gastrointestinal environment].

In an experiment with 4 x 12 piglets the probiotic effect of the Bacillus cereus strain FH 1457 S on the performance and changes in the gastrointestinal tract was studied. Beside a negative control the feed was supplemented with 10(7), 10(8) and 10(9) CFU Bacillus cereus/kg. The experiment included two feeding periods, whereby in the first a prestarter and in the second a piglet rearing feed was administrated ad libitum with the animals kept individually in cages. Over the whole feeding trial the piglets with the additions of 10(7), 10(8) und 10(9) CFU Bacillus cereus/kg feed showed higher daily weight gains of 9.5%, 11.1% and 8.6% with an increased feed consumption of 11.1%, 11.0% and 7.9%. The feed conversion rate was not influenced by the additions. In the first period daily weight gains were increased by 8.9%, 14.7% and 7.8%, the feed consumption in this period was 10.2%, 12.1% and 7.2% higher than in the control group. The addition of 10(8) CFU Bacillus cereus had a significantly higher daily weight gain in the first period compared to the control and the feed conversion rate was improved by 2.4%. In the second period the daily weight gains were increased by 9.9%, 8.9% and 9.1%, the feed consumption by 11.4%, 10.5% and 8.8% because of Bacillus cereus addition to the feed. The frequency of diarrhea was not influenced by the treatments. In each Bacillus cereus dosage the concentrations of ammonia, lactate and volatile fatty acids in small intestine contents were mostly diminished. In caecum contents the concentrations of acetic and propionic acid were decreased in the groups supplemented with Bacillus cereus.

Ammonia↗
PubMed1993

[The nutritive effect of Bacillus cereus as a probiotic in the raising of piglets. 2. Effect and microbial count, composition and resistance determination of gastrointestinal and fecal microflora].

After a feeding trial of 42 days with 4 x 12 piglets 4 x 6 piglets were slaughtered and the influence of spores of the Bacillus cereus strain FH 1457 S added to the feed on the microorganism counts of Lactobacillus/Bifidobacterium, Eubacteria, Bacteroidaceae, E. coli, Enterococcus and passants (Bacillus cereus) in duodenum, jejunum, ileum, caecum and colon was investigated. Beside a negative control the feed was supplemented with 10(7), 10(8) and 10(9) CFU Bacillus cereus/kg. The feeding trial included two periods each of 21 days. In each period faeces was collected from all animals and the influence on the microorganism counts and also the frequency of resistance of the E. coli and Enterococcus germs against selected antibiotics and chemotherapeutics investigated. The addition of 10(8) CFU Bacillus cereus decreased E. coli counts in duodenum and jejunum, however increased them in ileum, caecum and colon. In the highest dosage the counts of Bacillus cereus in duodenum, caecum, colon and faeces were significantly higher. The addition of Bacillus cereus reduced the counts of E. coli and Enterococcus in faeces in the first period. In the second period the dosage 10(8) CFU showed significant increased counts of E. coli and the sums of microorganisms of the main and satellite flora. The additions had no influence on the frequency of resistance of E. coli and enterococci against the tested antibiotics and synthetic chemotherapeutics. From the addition of Bacillus cereus in this case a selection of factors influencing resistance is not to be expected.

Animal Feed↗
PubMed2006

Inhibitory effects of spice essential oils on the growth of Bacillus species.

A series of essential oils of 11 Turkish plant spices [black thyme, cumin, fennel (sweet), laurel, marjoram, mint, oregano, pickling herb, sage, savory, and thyme], used in foods mainly for their flavor, aromas, and preservation, in herbal tea, in alternative medicines, and in natural therapies, were screened for antibacterial effects at 1:50, 1:100, 1:250, and 1:500 dilutions by the paper disc diffusion method against six Bacillus species (Bacillus amyloliquefaciens ATCC 3842, Bacillus brevis FMC 3, Bacillus cereus FMC 19, Bacillus megaterium DSM 32, Bacillus subtilis IMG 22, and B. subtilis var. niger ATCC 10). All of the tested essential oils (except for cumin) showed antibacterial activity against one or more of the Bacillus species used in this study. Generally, the essential oils at 1:50 and 1:100 levels were more effective. Only one essential oil (laurel) was not found effective against the tested bacteria. The bacterium most sensitive to all of the spice essential oils was B. amyloliquefaciens ATCC 3842. Based on the results of this study, it is likely that essential oils of some spices may be used as antimicrobial agents to prevent the spoilage of food products.

Anti-Bacterial Agents↗
PubMed1991

Numerical analysis and DNA base compositions of some thermophilic Bacillus species.

Morphological, physiological, and biochemical characteristics and the DNA base compositions of 133 thermophilic Bacillus strains were determined. A total of 54 of these strains were received as identified species (mainly Bacillus stearothermophilus, Bacillus coagulans, Bacillus brevis, and Bacillus licheniformis) from international culture collections, and 79 newly isolated strains, which were isolated mainly from sugar diffusion juices of Italian plants, were also examined. Numerical taxonomy techniques (simple matching coefficient and unweight pair grouping using the mathematical average) and DNA G + C values showed that the strains aggregated into nine clusters. Both B. licheniformis and B. brevis were well separated from the other organisms. B. stearothermophilus and B. coagulans were confirmed as separate clusters and exhibited greater heterogeneity than previously shown. The B. stearothermophilus strains clustered into four groups, three of which have been recognized previously by other authors; the members of the fourth group had distinctive characteristics, including considerable biochemical inertness, an inability to grow at temperatures greater than 60 degrees C, and a high G + C content. Within the B. coagulans cluster the strains with characteristics very similar to those of the new species Bacillus smithii clustered together. However, the remaining strains were still clearly separated into two groups; one of these groups was considered B. coagulans sensu stricto, and the other was distinguished by morphological and biochemical criteria, such as spores which do not swell the sporangia, utilization of citrate, a higher proteolytic activity, and acidification of some carbohydrates. Our results were confirmed by comparing them with distinctive characteristics of recently described thermophilic Bacillus species.

Aerobiosis↗
PubMed1994

Phylogenetic diversity in the genus Bacillus and comparative ribosomal protein AT-L30 analyses of the genus Thermoactinomyces and relatives.

The ribosomal L30 proteins from strains of 27 species belonging to the genera Bacillus, Escherichia, Staphylococcus, Lactobacillus, Leuconostoc and Thermoactinomyces were analysed, together with AT-L30 proteins from selected actinomycetes. The results of partial amino acid sequencing of L30 preparations revealed that the members of the genera Escherichia, Staphylococcus and Thermoactinomyces were homogeneous within each genus. In contrast, phylogenetic diversity existed in the genus Bacillus, which contained at least four clusters. One cluster that contained Bacillus subtilis and Bacillus stearothermophilus was more closely related to the genus Staphylococcus than to members of the other three Bacillus clusters. Members of the genus Thermoactinomyces were most closely related to the 'Bacillus subtilis cluster', but less related to the other three Bacillus clusters. A distant phylogenetic relationship was detected between the genus Thermoactinomyces and its morphological relative, Thermomonospora.

Amino Acid Sequence↗
PubMed2003

Bacillus barbaricus sp. nov., isolated from an experimental wall painting.

In a project concerning bacterial colonization of experimental wall paintings, a large number of isolates have been acquired with high similarities in their whole-cell protein patterns obtained after SDS-PAGE. Of this group, four strains, designated V2-BIII-A2(T), V2-BI-A9, V2-BI-04 and V2-BII-A8, were chosen for further characterization. Banding patterns obtained after ERIC-PCR were barely distinguishable among these four strains, indicating their affiliation within a single species. The isolates also displayed nearly identical biochemical and physiological features. The chemotaxonomic characteristics, including polar lipids, quinone systems, cell-wall diamino acid composition and fatty acid profiles, were in good agreement with those of numerous previously described Bacillus species. 16S rDNA analysis of strain V2-BIII-A2(T) showed that this bacterium belongs to the genus Bacillus, with highest sequence similarities to Bacillus megaterium (94.6%), Bacillus flexus (94.4%) and the alkaliphilic Bacillus cohnii (94.2%). Based on almost identical biochemical, physiological and chemotaxonomic traits, ERIC-PCR-generated genomic fingerprints and comparative 16S rDNA sequence analysis, it is demonstrated that the four isolates represent a novel species of the genus Bacillus, for which the name Bacillus barbaricus sp. nov. is proposed. The type strain is V2-BIII-A2(T) (= CCM 4982(T) = DSM 14730(T)).

Austria↗
PubMed2005

Bacillus alveayuensis sp. nov., a thermophilic bacterium isolated from deep-sea sediments of the Ayu Trough.

Two thermophilic, spore-forming strains, TM1(T) and TM5, were isolated from deep-sea sediment (4000 m below sea level) of the Ayu Trough in the western Pacific Ocean. Cells of the two strains were Gram-positive, motile and rod-shaped. Their spores were ellipsoidal, subterminal to terminal and occurred in swollen sporangia. The two strains grew at temperatures up to 65 degrees C and in the pH range 6.5-9.0. The NaCl concentration for optimal growth was 3.0 % (w/v) and growth was inhibited by 5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains TM1(T) and TM5 belonged to the genus Bacillus, and that strain TM1(T) was most closely related to Bacillus aeolius DSM 15084(T) (96.7 %), Bacillus smithii DSM 4216(T) (96.1 %), Bacillus methanolicus NCIMB 13113(T) (95.8 %) and Bacillus pallidus DSM 3670(T) (95.7 %). Between the 16S rRNA gene sequences of strains TM1(T) and TM5 there were only three nucleotide differences, implying that the two strains were of the same species. The cellular fatty acid profiles of the two strains were also very similar, with iso-C(15 : 0), iso-C(16 : 0), C(16 : 0), iso-C(17 : 0) and anteiso-C(17 : 0) as the major components. The G + C content of strain TM1(T) was 38.7 %. On the basis of phenotypic and molecular data, strains TM1(T) and TM5 represent a novel species of the genus Bacillus, for which the name Bacillus alveayuensis sp. nov. is proposed. The type strain is TM1(T) (= KCTC 10634(T) = JCM 12523(T)).

Bacillus↗
PubMed1989

The Na+-motive terminal oxidase activity in an alkalo- and halo-tolerant Bacillus.

An alkalo- and halo-tolerant aerobic microorganism has been isolated which, according to microbiological analysis data and the ribosomal 5S RNA sequence, is a Bacillus similar, but not identical, to B. licheniformis and B. subtilis. The microorganism, called Bacillus FTU, proved to be resistant to the protonophorous uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). The fast growth of Bacillus FTU in the presence of CCCP was shown to require a high Na+ concentration in the medium. A procedure was developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only when an exogenous respiratory substrate was added. The exhausted cells were found to oxidize ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) in a cyanide-sensitive fashion. The ascorbate oxidation was coupled to the uphill Na+ extrusion which was stimulated by CCCP and a penetrating weak base, diethylamine, as well as by valinomycin with or without diethylamine. Operation of the Bacillus FTU terminal oxidase resulted in the generation of a delta psi which, in the Na+ medium, was slightly decreased by CCCP and strongly decreased by CCCP + diethylamine. In the K+ medium, CCCP discharged delta psi even without diethylamine. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na+ medium and sensitive to CCCP in the K+ medium as if Na+- and H+-coupled oxidative phosphorylations were operative in the Na+ and K+ media, respectively. Inside-out subcellular vesicles of Bacillus FTU were found to be competent in the Na+ uptake supported by oxidation of ascorbate + TMPD or diaminodurene. CCCP or valinomycin + K+ increased the Na+ uptake very strongly. The process was completely inhibited by cyanide or monensin, the former, but not the latter, being inhibitory for respiration. The data obtained indicate that in Bacillus FTU there is not only H+-motive but also Na+-motive terminal oxidase activity.

Ascorbic Acid↗
PubMed2006

Composition of bacillus species in aerosols from 11 U.S. cities.

A PCR-based heteroduplex assay was used to determine the presence and composition of Bacillus species in 11,059 Environmental Protection Agency PM2.5 aerosol samples from 11 U.S. cities. The assay differentiated three groups: Type A containing Bacillus anthracis and very closely related, often pathogenic, Bacillus cereus and Bacillus thuringiensis strains; Type B containing other B. cereus and B. thuringiensis strains; and a third group of more-distantly related Bacillus species. Eight of the 11 cities were positive for Bacillus species in 50% or more of the samples, and the percent of aerosol samples that contained the HD Type A group ranged from 3% to 32%. Cities from the eastern half of the United States generally contained a higher frequency and broader diversity of Bacillus species than the western half of the United States. Positive samples were detected throughout the year. These results have implications for pathogen detection in environmental samples, understanding the natural evolution of new pathogenic strains, and incidence of infection caused by strains of the B. cereus subgroup.

Aerosols↗
PubMed2001

Molecular characterization and identification of Bacillus clausii Strains marketed for use in oral bacteriotherapy.

A substantial number of Bacillus species have been marketed for use in oral bacteriotherapy because of their purported ability to prevent or treat various gastrointestinal disorders. Recently, some of the Bacillus strains in Enterogermina, which is made up of aqueous suspensions of viable Bacillus spores, have been partially characterized and aligned with members of the Bacillus alcalophilus subgroup rather than with Bacillus subtilis, as previously reported. With a view toward verifying the original taxonomic position of the Enterogermina strains, we catalogued both phenotypic and genotypic traits exhibited by the four Bacillus strains isolated from the spore mixtures found in original commercial preparations dated 1975 and 1984 and commercial preparations now being propagated industrially. Analyses of physiological and biochemical traits, complete 16S rRNA gene sequences, DNA-DNA reassociation, tRNA intergenic spacer length polymorphism, single-strand conformation polymorphism of PCR-amplified spacer regions of tRNA genes, and randomly amplified polymorphic DNA led to the finding that all of the Enterogermina strains belong to a unique genospecies, which is unequivocally identified as the alkalitolerant species Bacillus clausii. Moreover, we provide evidence that in contrast to several reference strains of B. clausii, the strains constituting Enterogermina are characterized by a notable low level of intraspecific genome diversity and that each strain has remained the same for the last 25 years.

Administration, Oral↗
PubMed2001

Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae.

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.

Animals↗
PubMed1974

Homologous and hybrid complexes of anthranilate synthase from Bacillus species.

The subunits of anthranilate synthase were separated and partially purified by Sephadex G-100 gel filtration from the following six species of Bacillus: Bacillus subtilis, Bacillus licheniformis, Bacillus alvei, Bacillus coagulans, Bacillus pumilus, and Bacillus mascerans. Our data suggest that the enzyme from B. alvei is unique among these species. First, the anthranilate synthase complexes are readily dissociated during gel filtration in the absence of glutamine into a large component (aminotransferase), subunit E, and a small component subunit X (glutamine-binding protein), whereas a higher salt concentration is required to dissociate the complex from B. alvei. Second, the aminotransferase activity from all six species is stimulated by glycerol and inhibited by tryptophan; however, only the large component from B. alvei is stimulated by 2-mercaptoethanol. Finally, the large component can be titrated with the small component to yield a complex which can utilize glutamine as a substrate (amidotransferase). The homologous complexes have an amidotransferase to aminotransferase ratio of 1.4 to 2.3, but the B. alvei complex has a ratio of 0.9. Except for complexes that involve the large component from B. alvei, hybrid complexes can be formed which have ratios as good as the homologous complexes. These data are consistent with the hypothesis that B. alvei is unique among the bacilli with respect to some enzymes in the aromatic amino acid biosynthetic pathway.

Amides↗