Histidine utilization by Alcaligenes eutrophus: Regulation of histidase formation under heterotrophic conditions of growth

Histidine supported good growth of Alcaligenes eutrophus strain H 16 as a nitrogen source, but only poor growth as a carbon and energy source. The facultative chemolithoautotrophic bacterium was also able to utilize urocanic acid, the first intermediate of histidine catabolism. The products of histidine degradation were ammonium, formate and glutamate. Three enzymes of the pathway, histidase, urocanase and formiminoglutamate hydrolase, were present in histidine-grown cells. Two types of spontaneous mutants, derived from the wild type, were characterized by an increased growth rate on histidine. One of these types was found to produce histidase constitutively and at a higher activity compared with the parental strain. The second type of mutant had apparently gained an improved histidine uptake system, which is supposed to be growth rate-limiting in the wild type. From the physiological studies the conclusion was drawn that the control of histidine-degrading enzymes is based on induction by urocanate and catabolite repression by carbon sources supporting fast growth, such as succinate or pyruvate. Ammonium was found not to affect catabolite repression, however, we obtained evidence that histidine uptake is subject to a nitrogen control.Abbreviation CTAB hexadecyltrimethylammonium bromide     

Evidence for carrier-mediated,energy-dependent uptake of urea in some bacteria

Evidence for the existence of an energy-dependent urea permease was found for Alcaligenes eutrophus H16 and Klebsiella pneumoniae M5a1 by studying uptake of ¹⁴C-urea. Since intracellular urea was metabolized immediately, uptake did not result in formation of an urea pool. Evidence is based on observations that the in vivo urea uptake and in vitro urease activity differ significantly with respect to kinetic parameters, temperature optimum, pH optimum, response towards inhibitors and regulation. The K _m for urea uptake was 15–20 times lower (38 M and 13 M urea for A. eutrophus and K. pneumoniae, respectively) than the K _m of urease for urea (650 M and 280 M urea), the activity optimum for A. eutrophus was at pH 6.0 and 35°C for the uptake and pH 9.0 and 65°C for urease. Uptake but not urease activity in both organisms strongly decreased upon addition of inhibitors of energy metabolism, while in K. pneumoniae, potent inhibitors of urease (thiourea and hydroxyurea) did not affect the uptake process. Significant differences in the uptake rates were observed during growth with different nitrogen sources (ammonia, nitrate, urea) or in the absence of a nitrogen source; this suggested that a carrier is involved which is subject to nitrogen control. Some evidence for the presence of an energy-dependent uptake of urea was also obtained in Pseudomonas aeruginosa DSM 50071 and Providencia rettgeri DSM 1131, but not in Proteus vulgaris DSM 30118 and Bacillus pasteurii DSM 33.Non-standard abbreviations CCCP Carbonylcyanide-m-chlorphenylhydrazone - DCCD dicyclohexylcarbodiimide - DNP 2,4-dinitrophenole     

Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D

Abstract Poly(3-hydroxybutyric acid) granules, which harbored only four major granule-associated proteins as revealed by SDS polyacrylamide gel electrophoresis, were isolated from crude cellular extracts of Chromatium vinosum D by centrifugation in a linear sucrose gradient. N-Terminal amino acid sequence determination identified two proteins of M _r 41 000 and M _{r 40 000} as the phaE _Cv and phaC _Cv translational products, respectively, of C. vinosum D. In a previous study it was shown that both proteins are required for the expression opf poly(3-hydroxyalkanoic acid) synthase activity. The N-terminus of the third protein ( M _r 17 000) exhibited no homology to other proteins. Lysozyme, which was during purification of the granules, exhibited a strong affinity to PHB granules and was identified as the fourth protein enriched with the granules.     

Anaerobic utilization of essential oils bydenitrifying bacteria

Plant volatile organic compounds are a major carbonsource in nature. We studied the degradability ofthese substances by anaerobic microorganisms inenrichment cultures with representative essential oilsas organic substrates and nitrate as electronacceptor. Lemon and pine needle oil supportedmicrobial growth in the presence of pure oil, whereasparsley seed, camphor, sage, fennel, and mint oilsupported growth only when the essential oils weredissolved in an overlying phase of2,2,4,4,6,8,8-heptamethylnonane. Thyme oil did notsupport denitrification. Analyses of the microbiallydegraded oils revealed the disappearance ofmonoterpenes, of several monoterpenoids, and ofmethoxy-propenyl-benzenes, including apiole andmyristicin. Most-probable-number determinations fordenitrifying communities in sewage sludge and forestsoil yielded 10⁶ to 10⁷monoterpene-utilizing cells ml^-1, representing0.7 to 100% of the total cultivablenitrate-reducing microorganisms. The utilization ofessential oils together with the common occurrence ofthis metabolic trait are indications for anenvironmentally important, but currently unexploredanaerobic turnover of plant volatile organic compoundsin soil.     

ε-Caprolactam-degradation by Alcaligenes faecalis for bioremediation of wastewater of a nylon-6 production plant

Alcaligenes faecalis G utilized 95–97% of 5–15 g -caprolactam l^–1 in 24–48 h over a pH range of 6–8.5 and at 23–40 °C, without complex nutrient requirement. In the absence of KH₂PO₄ and K₂HPO₄/MgSO₄ in the medium, only 7.6% and 0.2% of 10 g caprolactam l^–1 was utilized, respectively. The chemical oxygen demand (COD) of the wastewater of nylon-6 plant was mainly due to its caprolactam content. A. faecalis G decreased the caprolactam content and COD of the wastewater by 80–90% of the original in spite of the wastewater having higher caprolactam content (3600 mg l^–1) and COD (7700 mg l^–1) than those of any of the previous reports.     

The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Alcaligenes eutrophus . Incorporation of [1-¹⁴C]-acetyl-CoA into poly-3-hydroxybutyrate (PHB) by systems reconstituted from purified preparations of either 3-ketothiolase, AcAc-CoA reductase and PHB synthase, occurred only when NADPH-AcAc-CoA reductase was present. The NADH reductase was active with all of the d (−)- and l (+)-3-hydroxyacyl-CoA substrates tested (C₄-C₁₀), whereas the NADPH reductase was only active with d (−)-3-hydroxyacyl-CoAs (C₄-C₆). The products of AcAc-CoA reduction by the NADH- and NADPH-linked enzymes were l (+)-3-hydroxybutyryl-CoA and d (−)-3-hydroxybutyryl-CoA, respectively. The NADH-linked enzyme had an M _r of 150,000 (containing identical M _r 30,000 sub-units) and the NADPH-linked enzyme appeared to be a tetramer ( M _r 84,000) with identical sub-units ( M _r 23,000). K _m^app values of 22 μM and 5 μM for AcAc-CoA and 13 μM (NADH) and 19 μM (NADPH) for the coenzymes were determined for the NADH- and NADPH-linked enzymes, respectively.     

Metabolism of 2-chloro-4-methylphenoxyacetate by Alcaligenes eutrophus JMP 134

2-Chloro-4-methylphenoxyacetate is not a growth substrate for Alcaligenes eutrophus JMP 134 and JMP 1341. It is, however, being transformed by enzymes of 2,4-dichlorophenoxyacetic acid metabolism to 2-chloro-4-methyl-cis, cis-muconate, which is converted by enzymatic 1,4-cycloisomerization to 4-carboxymethyl-2-chloro-4-methylmuconolactone as a dead end metabolite. Chemically, only 3,6-cycloisomerization occurs, giving rise to both diastereomers of 4-carboxychloromethyl-3-methylbut-2-en-4-olide. Those lactones harbonring a chlorosubstituent on the 4-carboxymethyl side chain were surprisingly stable under physiological as well as acidic conditions.     

Conversion of 2-chloromaleylacetate in Alcaligenes eutrophus JMP134

2,4-Dichlorophenoxyacetate (2,4-D) in Alcaligenes eutrophus JMP134 (pJP4) is degraded via 2-chloromaleylacetate as an intermediate. The latter compound was found to be reduced by NADH in a maleylacetate reductase catalyzed reaction. Maleylacetate and chloride were formed as products of 2-chloromaleylacetate reduction, the former being funnelled into the 3-oxoadipate pathway by a second reductive step. There was no indication for an involvement of a pJP4-encoded enzyme in either the reduction or the dechlorination reaction.Abbreviations 2,4-D 2,4-dichlorophenoxyacetate