Degradation of hydroxyhydroquinone by the strictly anaerobic fermenting bacterium Pelobacter massiliensis sp. nov.

A new rod-shaped, gram-negative, non-sporeforming, strictly anaerobic bacterium (strain HHQ7) was enriched and isolated from marine mud samples with hydroxyhydroquinone (1,2,4-trihydroxybenzene) as sole substrate. Strain HHQ7 fermented hydroxyhydroquinone, pyrogallol (1,2,3-trihydroxybenzene), phloroglucinol (1,3,5-trihydroxybenzene) and gallic acid (3,4,5-trihydroxybenzoate) to 3 mol acetate (plus 1 mol CO₂ in the case of gallic acid) per mol of substrate. Resorcinol accumulated intermediately during growth on hydroxy-hydroquinone. No other aliphatic or aromatic substrates were utilized. Sulfate, sulfite, sulfur, nitrate, and fumarate were not reduced with hydroxyhydroquinone as electron donor. The strain grew in sulfide-reduced mineral medium supplemented with 7 vitamins. The DNA base ratio was 59% G+C. Strain HHQ7 is classified as a new species of the genus Pelobacter, P. massiliensis. Experiments with dense cell suspensions of hydroxyhydroquinone-and pyrogallol-grown cells showed different kinetics of hydroxyhydroquinone and pyrogallol degradation, as well as different patterns of resorcinol accumulation, indicating that these substrates are metabolized by different transhydroxylation reactions.     

Anaerobic degradation of 1,3-propanediol by sulfate-reducing and by fermenting bacteria

Three strains of strictly anaerobic Gram-negative, non-sporeforming, motile bacteria were enriched and isolated from freshwater sediments with 1,3-propanediol as sole energy and carbon source. Strain OttPdl was a sulfate-reducing bacterium which grew also with lactate, ethanol, propanol, butanol, 1,4-butanediol, formate or hydrogen plus CO₂, the latter only in the presence of acetate. In the absence of sulfate, most of these substrates were fermented to the respective fatty acids in syntrophic cooperation with Methanospirillum hungatei. Sulfur, thiosulfate, or sulfite were reduced, nitrate not. The other two isolates degraded propanediol only in coculture with Methanospirillum hungatei. Strain OttGlycl grew in pure culture with acetoin and with glycerol in the presence of acetate. Strain WoAcl grew in pure culture only with acetoin. Both strains did not grow with other substrates, and did not reduce nitrate, sulfate, sulfur, thiosulfate or sulfite. The isolates were affiliated with the genera Desulfovibrio and Pelobacter. The pathways of propanediol degradation and the ecological importance of this process are discussed.     

Linear alkanesulfonates as carbon and energy sources for gram-positive and gram-negative bacteria

Several bacteria from soil and rainwater samples were enriched and isolated with propanesulfonate or butanesulfonate as sole carbon and energy source. Most of the strains isolated utilized nonsubstituted alkanesulfonates with a chain length of C3–C6 and the substituted sulfonates taurine and isethionate as carbon and energy source. A gram-positive isolate, P40, and a gram-negative isolate, P53, were characterized in more detail. Phylogenetic analysis grouped strain P40 within group IV of the genus Rhodococcus and showed a close relationship with Rhodococcus opacus. After phylogenetic and physiological analyses, strain P53 was identified as Comamonas acidovorans. Both bacteria also utilized a wide range of sulfonates as sulfur source. Strain P40, but not strain P53, released sulfite into the medium during dissimilation of sulfonated compounds. Cell-free extracts of strain P53 exhibited high sulfite oxidase activity [2.34 U (mg protein)^–1] when assayed with ferricyanide, but not with cytochrome c. Experiments with whole-cell suspensions of both strains showed that the ability to dissimilate 1-propanesulfonate was specifically induced during growth on this substrate and was not present in cells grown on propanol, isethionate or taurine. Whole-cell suspensions of both strains accumulated acetone when oxidizing the non-growth substrate 2-propanesulfonate. Strain P40 cells also accumulated sulfite under these conditions. Stoichiometric measurements with 2-propanesulfonate as substrate in oxygen electrode experiments indicate that the nonsubstituted alkanesulfonates were degraded by a monooxygenase. When strain P53 grew with nonsubstituted alkanesulfonates as carbon and energy source, cells expressed high amounts of yellow pigments, supporting the proposition that an oxygenase containing iron sulfur centres or flavins was involved in their degradation. Received: 21 December 1998 / Accepted: 18 March 1999     

Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and p-xylene by two Pseudomonas isolates

Two Pseudomonas species (designated strains B1 and X1) were isolated from an aerobic pilot-scale fluidized bed reactor treating groundwater containing benzene, toluene, and p-xylene (BTX). Strain B1 grew with benzene and toluene as the sole sources of carbon and energy, and it cometabolized p-xylene in the presence of toluene. Strain X1 grew on toluene and p-xylene, but not benzene. In single substrate experiments, the appearance of biomass lagged the consumption of growth substrates, suggesting that substrate uptake may not be growth-rate limiting for these substrates. Batch tests using paired substrates (BT, TX, or BX) revealed competitive inhibition and cometabolic degradation patterns. Competitive inhibition was modeled by adding a competitive inhibition term to the Monod expression. Cometabolic transformation of nongrowth substrate (p-xylene) by strain B1 was quantified by coupling xylene transformation to consumption of growth substrate (toluene) during growth and to loss of biomass during the decay phase. Coupling was achieved by defining two transformation capacity terms for the cometabolizing culture: one that relates consumption of growth substrate to the consumption of nongrowth substrate, and second that relates consumption of biomass to the consumption of nongrowth substrate. Cometabolism increased decay rates, and the observed yield for strain B1 decreased in the presence of p-xylene. (c) 1993 Wiley & Sons, Inc.     

The ssu locus plays a key role in organosulfur metabolism in Pseudomonas putida S-313

Pseudomonas putida S-313 can utilize a broad range of aromatic sulfonates as sulfur sources for growth in sulfate-free minimal medium. The sulfonates are cleaved monooxygenolytically to yield the corresponding phenols. miniTn5 mutants of strain S-313 which were no longer able to desulfurize arylsulfonates were isolated and were found to carry transposon insertions in the ssuEADCBF operon, which contained genes for an ATP-binding cassette-type transporter (ssuABC), a two-component reduced flavin mononucleotide-dependent monooxygenase (ssuED) closely related to the Escherichia coli alkanesulfonatase, and a protein related to clostridial molybdopterin-binding proteins (ssuF). These mutants were also deficient in growth with a variety of other organosulfur sources, including aromatic and aliphatic sulfate esters, methionine, and aliphatic sulfonates other than the natural sulfonates taurine and cysteate. This pleiotropic phenotype was complemented by the ssu operon, confirming its key role in organosulfur metabolism in this species. Further complementation analysis revealed that the ssuF gene product was required for growth with all of the tested substrates except methionine and that the oxygenase encoded by ssuD was required for growth with sulfonates or methionine. The flavin reductase SsuE was not required for growth with aliphatic sulfonates or methionine but was needed for growth with arylsulfonates, suggesting that an alternative isozyme exists for the former compounds that is not active in transformation of the latter substrates. Aryl sulfate ester utilization was catalyzed by an arylsulfotransferase, and not by an arylsulfatase as in the related species Pseudomonas aeruginosa.     

Regulation of the utilization of glucose and aromatic substrates in four strains of Pseudomonas putida

During the oxidation of various mixtures of glucose and aromatic substrates by four strains of Pseudomonas putida, diauxic growth was not observed. Strain A3.12 grew faster on benzoate than on glucose, whereas three other strains showed faster growth on glucose than on the aromatic test substrates. Growth rates on mixtures of glucose and aromatics were intermediate between those on the single substrates.The presence of glucose in media containing aromatic substrates accelerated in the bacteria the appearance of the ability to oxidize aromatic substrates. During growth of the organisms on binary mixtures of aromatics, simultaneous utilization of these compounds occurred, the utilization ratio depending on the quality of the compounds as carbon and energy sources. Addition of glucose to dual aromatic substrate media greatly increased the utilization ratio in favour of the better aromatic substrate.With decreasing concentration of glucose in relation to that of aromatic substrates, the rate of carbon assimilation from glucose increased. Enzymological and radiochemical studies demonstrated that even in the presence of an excess of aromatic substrates, glucose was exclusively catabolized via the 2-keto-3-deoxy-6-phosphogluconate pathway. In contrast, the rate of carbon assimilation from ¹⁴C-ring-labelled benzoate and anisate was unaffected by the presence of an excess of glucose.Abbreviations KDPG 2-keto-3-deoxy-6-phosphogluconate - PP pentose-phosphate - OD optical density     

Spectroscopic characterization and identification of Pseudomonas fluorescens mediated metabolic products of Acid Yellow-9

Pseudomonas flourescens NCIM 2100 was obtained from NCL, Pune, India, that was adapted to growth on 4 amino 1-1 azo benzene 3,4-Disulfonic acid. This strains was tested by UV, ¹H NMR, and IR spectroscopy for its ability to degrade the dye which resulted in to sulfonated analogs namely p-amino benzene sulfonic acid sodium salt and 2,4 diamino benzene sulfonic acid sodium salt. These compound further changed to either 2,4 dihydroxy benzene sulfonic acid sodium salt or 2 amino 4 hydroxy benzene sulfonic acid sodium salt, or 4 amino 2 hydroxy benzene sulfonic acid sodium salt. These breakdown compounds were non toxic in nature.     

Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12

Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g. sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist.     

Utilization of Halogenated Benzenes, Phenols, and Benzoates by Rhodococcus opacus GM-14

Strain GM-14 was isolated by selective enrichment from contaminated soil with chlorobenzene as the sole source of carbon and energy. It utilizes an exceptionally wide spectrum of haloaromatic substrates. It is a gram-positive, weakly acid-fast actinomycete, with a morphological cycle from cocci and short rods to long rods and branched filaments; it grew optimally at 28(deg)C; and it tolerated 5% NaCl in rich medium. The chemotaxonomic characteristics, the diagnostic biochemical tests, the whole-cell fatty acid composition, and 16S rDNA analysis were consistent with Rhodococcus opacus. R. opacus GM-14 grew on 48 of 117 different aromatic and haloaromatic compounds. It utilized phenol at concentrations up to 1.2 g/liter, 3- and 4-methylphenols up to 0.5 g/liter, 2- and 4-chlorophenols up to 0.25 g/liter, and 3-chlorophenol up to 0.1 g/liter. It grew in saturated aqueous solutions of benzene, chlorobenzene, and 1,3- and 1,4-dichlorobenzene (up to 13, 3, 0.5, and 0.5 g/liter, respectively). The specific growth rate of strain GM-14 on phenol and 3- and 4-chlorophenols in batch culture was 0.27 to 0.29 h(sup-1), and that on benzene and chlorobenzene was similar to the rate on fructose, i.e., 0.2 h(sup-1). The growth yield on benzene and on chlorobenzene (<=0.4 g liter(sup-1)) was 40 to 50 g (dry weight) per mol of substrate consumed, equalling 8 g of dry weight biomass per mol of substrate carbon, similar to that obtained on acetate. During growth of strain GM-14 on chlorobenzene, 1,3-dichlorobenzene, and all isomers of monochlorophenol, stoichiometric amounts of chloride were released, and 50% of the stoichiometric amount was released from 1,4-dichlorobenzene.     

Anaerobic degradation of aniline and dihydroxybenzenes by newly isolated sulfate-reducing bacteria and description of Desulfobacterium anilini

A new, rod-shaped, Gram-negative, non-sporing sulfate reducer (strain Ani1) was enriched and isolated from marine sediment with aniline as sole electron donor and carbon source. The strain degraded aniline completely to CO2 and NH3 with stoichiometric reduction of sulfate to sulfide. Strain Ani1 also degraded aminobenzoates and further aromatic and aliphatic compounds. The strain grew in sulfide-reduced mineral medium supplemented only with vitamin B12 and thiamine. Cells contained cytochromes, carbon monoxide dehydrogenase, and sulfite reductase P582, but no desulfoviridin. Strain Ani1 is described as a new species of the genus Desulfobacterium D. anilini. Marine enrichments with the three dihydroxybenzene isomers led to three different strains of sulfate-reducing bacteria; each of them could grow only with the isomer used for enrichment. Two strains isolated with catechol (strain Cat2) or resorcinol (strain Re10) were studied in detail. Both strains oxidized their substrates completely to CO2, and contained cytochromes, carbon monoxide dehydrogenase, and sulfite reductase P 582. Desulfoviridin was not present. Whereas the rod-shaped catechol oxidizer (strain Cat2) was able to grow on 18 aromatic compounds and several aliphatic substrates, the coccoid resorcinol-degrading bacterium (strain Re10) utilized only resorcinol, 2,4-dihydroxybenzoate and 1,3-cyclohexanedion. These strains could not be affiliated with existing species of sulfate-reducing bacteria. A further coccoid sulfate-reducing bacterium (strain Hy5) was isolated with hydroquinone and identified as a subspecies of Desulfococcus multivorans. Most-probable-number enumerations with catechol, phenol, and resorcinol showed relatively large numbers (10(4)-10(6) per ml) of aryl compound-degrading sulfate reducers in marine sediment samples.     

The Development of Aerobic Spoilage Flora on Meat Stored at Chill Temperatures

In meat juice medium, aerobic spoilage bacteria utilized the following substrates in the order shown: Pseudomonos , glucose, amino acids, lactic acid; Acinetobacter , amino acids, lactic acid: Enterobacter , glucose, glucose-6-phosphate, amino acids; Microbacterium thermosphactum , glucose, glutamate. All the bacteria grew at their maximum rate utilizing the first and second substrates, but the growth rates declined when these were exhausted. The growth rate of Acinetobacter was reduced at pH 5·7 and below. All other species grew at their maximum rate within the pH range 5·5–7·0. On meat pseudomonads grew faster than the other species at all temperatures between 2° and 15°C. Interactions between any two species were observed only when one organism had attained its maximum cell density. Substrate exhaustion at the meat surface did not limit bacterial growth and it is suggested that the maximum cell density of aerobic spoilage cultures is determined by oxygen limitation of growth.     

Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU.

Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204-210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.     

Roles of ring-hydroxylating dioxygenases in styrene and benzene catabolism in Rhodococcus jostii RHA1

Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcus jostii RHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the ΔbphAa and etbAa1::cmrA etbAa2::aphII mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the β-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta- and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling.     

Catabolism of phenylpropionic acid and its 3-hydroxy derivative by Escherichia coli

A number of laboratory strains and clinical isolates of Escherichia coli utilized several aromatic acids as sole sources of carbon for growth. E. coli K-12 used separate reactions to convert 3-phenylpropionic and 3-(3-hydroxyphenyl)propionic acids into 3-(2,3-dihydroxyphenyl)propionic acid which, after meta-fission of the benzene nucleus, gave succinate, pyruvate, and acetaldehyde as products. Enzyme assays and respirometry showed that all enzymes of this branched pathway were inducible and that syntheses of enzymes required to convert the two initial growth substrates into 3-(2,3-dihydroxyphenyl)propionate are under separate control. E. coli K-12 also grew with 3-hydroxycinnamic acid as sole source of carbon; the ability of cells to oxidize cinnamic and 3-phenylpropionic acids, and hydroxylated derivatives, was investigated. The lactone of 4-hydroxy-2-ketovaleric acid was isolated from enzymatic reaction mixtures and its properties, including optical activity, were recorded.     

Growth rate and physiology of Yersinia enterocolitica; influence of temperature and presence of the virulence plasmid

The effect of temperature on the growth rate, protein pattern and fatty acid composition of Yersinia enterocolitica strain W22703 pYV⁺, its plasmidless isogenic derivative W22703 pYV^- and four recent field isolates was examined.
The growth rate was clearly influenced by presence or absence of the virulence plasmid: pYV^- strains grew consistently faster than pYV⁺ strains. This difference in growth rate was high at 30–35°C, moderate at 1–10°C and 25°C, but hardly significant at 15–20°C.
Increasing the growth temperature above 25°C resulted in the induction of the 220 kDa virulence plasmid-encoded Yop1 protein. In the 1–20°C range no obvious temperature- or plasmid-related differences in protein patterns could be detected.
The fatty acid composition showed a clear temperature-dependent change: with all strains the degree of saturation was low at 1°C and gradually increased with raising temperatures. All strains had similar fatty acid patterns, except one of the field isolates which showed aberrant C16 : 1 and cyclic fatty acid contents in the 5–25°C and 15°C ranges respectively. With strain W22703, the presence or absence of the virulence plasmid did not significantly alter the fatty acid pattern.     

Sulfonate-sulfur assimilation by yeasts resembles that of bacteria

Abstract Three sulfonates were tested for their ability to serve as nutrients for Hansenula wingei, Rhodotorula glutinis, Trigonopsis variabilis and Saccharomyces cerevisiae . Cysteate, taurine and isethionate, under aerobic conditions, could be utilized as sources of sulfur, although in some instances final cell yields were less than those obtained with an equimolar amount of sulfate-sulfur. Sulfonate assimilation by S. cerevisiae resembled that of bacteria (reported earlier by us) in several aspects: first, sulfate-S was used in preference to that of sulfonate, when both were present; second, mutants unable to use a source of sulfur because of deficiencies in ATP sulfurylase, adenylylsulfate kinase (APS kinase) or PAPS reductase were able to utilize sulfonates; and third, mutants deficient in sulfite reductase were unable to utilize sulfonates.     

Isolation and characterization of Desulfovibrio dechloracetivorans sp. nov., a marine dechlorinating bacterium growing by coupling the oxidation of acetate to the reductive dechlorination of 2-chlorophenol

Strain SF3, a gram-negative, anaerobic, motile, short curved rod that grows by coupling the reductive dechlorination of 2-chlorophenol (2-CP) to the oxidation of acetate, was isolated from San Francisco Bay sediment. Strain SF3 grew at concentrations of NaCl ranging from 0.16 to 2.5%, but concentrations of KCl above 0. 32% inhibited growth. The isolate used acetate, fumarate, lactate, propionate, pyruvate, alanine, and ethanol as electron donors for growth coupled to reductive dechlorination. Among the halogenated aromatic compounds tested, only the ortho position of chlorophenols was reductively dechlorinated, and additional chlorines at other positions blocked ortho dechlorination. Sulfate, sulfite, thiosulfate, and nitrate were also used as electron acceptors for growth. The optimal temperature for growth was 30 degrees C, and no growth or dechlorination activity was observed at 37 degrees C. Growth by reductive dechlorination was revealed by a growth yield of about 1 g of protein per mol of 2-CP dechlorinated, and about 2.7 g of protein per mole of 2,6-dichlorophenol dechlorinated. The physiological features and 16S ribosomal DNA sequence suggest that the organism is a novel species of the genus Desulfovibrio and which we have designated Desulfovibrio dechloracetivorans. The unusual physiological feature of this strain is that it uses acetate as an electron donor and carbon source for growth with 2-CP but not with sulfate.     

Transport and utilization of the biosynthetic intermediate shikimic acid in Escherichia coli.

Auxotrophic mutants of Escherichia coli W or K12 blocked before shikimic acid in the aromatic biosynthetic pathway grew poorly on shikimic acid as sole aromatic supplement. This poor growth response was correlated with a relatively poor ability to transport shikimic acid. If citrate was present in the growth medium (as it is in some commonly used basal media) the growth of some of the E. coli K12 mutants on shikimate was further reduced. Mutants were derived from pre-shikimate auxotrophs which grew rapidly on media containing shikimic acid. These derivatives all had an increased ability to transport shikimic acid. Thus, it is proposed that the growth on shikimate observed in the parent cells is restricted by their relatively poor uptake of shikimate from the medium and that this restriction may be removed by a mutation which enhances shikimate transport. Transduction analysis of the mutations which enhanced utilization and transport of shikimic acid by E. coli K12 strains indicated at least two classes. Class 1 was about 20% cotransduced with the histidine region of the E. coli K12 chromosome and appeared to be coincident with a known shikimate transport locus, shiA. Class 2 was not cotransduced with his. The locus (or loci) of this class is unknown. Kinetic measurements suggested that both classes had shikimate uptake systems derived from the wild-type system. Two class 1 mutants had increased levels of otherwise unaltered wild-type transport while one class 2 mutant had an altered Michaelis constant (Km) for shikimate transport.     

Characterization of anaerobic sulfite reduction by Salmonella typhimurium and purification of the anaerobically induced sulfite reductase.

Mutants of Salmonella typhimurium that lack the biosynthetic sulfite reductase (cysI and cysJ mutants) retain the ability to reduce sulfite for growth under anaerobic conditions (E. L. Barrett and G. W. Chang, J. Gen. Microbiol., 115:513-516, 1979). Here we report studies of sulfite reduction by a cysI mutant of S. typhimurium and purification of the associated anaerobic sulfite reductase. Sulfite reduction for anaerobic growth did not require a reducing atmosphere but was prevented by an argon atmosphere contaminated with air (less than 0.33%). It was also prevented by the presence of 0.1 mM nitrate, which argues against a strictly biosynthetic role for anaerobic sulfite reduction. Anaerobic growth in liquid minimal medium, but not on agar, was found to require additions of trace amounts (10(-7)M) of cysteine. Spontaneous mutants that grew under the argon contaminated with air also lost the requirement for 10(-7)M cysteine for anaerobic growth in liquid. A role for sulfite reduction in anaerobic energy generation was contraindicated by the findings that sulfite reduction did not improve cell yields, and anaerobic sulfite reductase activity was greatest during the stationary phase of growth. Sulfite reductase was purified from the cytoplasmic fraction of the anaerobically grown cysI mutant and was purified 190-fold. The most effective donor in crude extracts was NADH. NADPH and methyl viologen were, respectively, 40 and 30% as effective as NADH. Oxygen reversibly inhibited the enzyme. Two high-molecular-weight proteins separated by gel filtration (Mr 360,000 and 490,000, respectively) were required for maximal activity with NADH. Indirect evidence, including in vitro complementation experiments with a cysG mutant extract, suggested that the 360,000-Mr component contains siroheme and is the terminal reductase. This component was further purified to near homogeneity and was found to consist of a single subunit of molecular weight 67,500. The anaerobic sulfite reductase showed some resemblance to the biosynthetic sulfite reductase, but apparently it has a unique, as yet unidentified function.     

Isolation and characterization of a new denitrifying spirillum capable of anaerobic degradation of phenol

Two kinds of phenol-degrading denitrifying bacteria, Azoarcus sp. strain CC-11 and spiral bacterial strain CC-26, were isolated from the same enrichment culture after 1 and 3 years of incubation, respectively. Both strains required ferrous ions for growth, but strain CC-26 grew better than strain CC-11 grew under iron-limited conditions, which may have resulted in the observed change in the phenol-degrading bacteria during the enrichment process. Strain CC-26 grew on phenol, benzoate, and other aromatic compounds under denitrifying conditions. Phylogenetic analysis of 16S ribosomal DNA sequences revealed that this strain is most closely related to a Magnetospirillum sp., a member of the alpha subclass of the class Proteobacteria, and is the first strain of a denitrifying aromatic compound-degrading bacterium belonging to this group. Unlike previously described Magnetospirillum strains, however, this strain did not exhibit magnetotaxis. It grew on phenol only under denitrifying conditions. Other substrates, such as acetate, supported aerobic growth, and the strain exhibited microaerophilic features.