Peter Jurtshuk

Phylogeny of Marine Bacillus Isolates from the Gulf of Mexico

The phylogeny of 11 pigmented, aerobic, spore-forming isolates from marine sources was studied. Forty-two biochemical characteristics were examined, and a 16S rDNA sequence was obtained for each isolate. In a phylogenetic tree based on 16S sequencing, four isolates (NRRL B-14850, NRRL B-14904, NRRL B-14907, and NRRL B-14908) clustered with B. subtilis and related organisms; NRRL B-14907 was closely related to B. amyloliquefaciens. NRRL B-14907 and NRRL B-14908 were phenotypically similar to B. amyloliquefaciens and B. pumilus, respectively. Three strains (NRRL B-14906, NRRL B-14910, and NRRL B-14911) clustered in a clade that included B. firmus, B. lentus, and B. megaterium. NRRL B-14910 was closely related phenotypically and phylogenetically to B. megaterium. NRRL B-14905 clustered with the mesophilic round spore-producing species, B. fusiformis and B. sphaericus; the isolate was more closely related to B. fusiformis. NRRL B-14905 displayed characteristics typical of the B. sphaericus-like organisms. NRRL B-14909 and NRRL B-14912 clustered with the Paenibacillus species and displayed characteristics typical of the genus. Only NRRL B-14851, an unusually thin rod that forms very small spores, may represent a new Bacillus species.

Bacterial Terminal Oxidases

AbstractOxidases, as such, regardless of their source, represent a diverse and complex series of enzymes. What they have in common is the ability to react with molecular oxygen, activate it chemically (in a manner which is still not understood), and utilize the “activated atoms of oxygen” primarily as electron acceptors. Should the “activated oxygen atoms” be used directly for oxygenating substrate molecules, such as hydrocarbons, then according to the conventions used today, the enzyme is termed an oxygenase rather than an oxidase. The subject of oxygenases is far too complex to be reviewed in any detailed treatment of oxidases. All oxidases serve as electron acceptors for specific dehydrogenation reactions that are carried out by the multitude of dehydrogenases that are found in tissues as well as in bacteria. The major end product that results from the oxidase reaction is either H2O or H2O2. The oxidases can be (1) simple flavoprotein-containing enzymes, such as the glucose oxidase or the D- and L-amin...

PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics.

The sequence of the major portion of aBacillus cycloheptanicus strain SCHT 16S rRNA gene is reported. This sequence suggests thatB. cycloheptanicus is genetically quite distinct from traditionalBacillus strains (e.g.,B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.

Characterization of the highly active isocitrate (NADP+) dehydrogenase of Azotobacter vinelandii.

The highly active isocitrate dehydrogenase [threo-ds-isocitrate:NADP oxidoreductase (decarboxylating), EC 1.1.1.42] was isolated and characterized from a clarified sonic cell-free extract of Azotobacter vinelandii Strain O. The purification was achieved by using a combination of (NH4)2SO4 precipitation and preparatory disc electrophoresis. Analytical disc electrophoresis studies revealed that the purified enzyme was a single protein component having an isoelectric point of 6.1. The molecular weight of the A. vinelandii isocitrate dehydrogenase was 78 000 and amino acid analyses showed that 3 half-cystine residues were present per molecule of enzyme. The enzyme possessed an exceptionally high νmax value. It reduced 722 μmoles of NADP+ per min per mg protein at 37°; the calculated turnover number was 56 000. The apparent Km for the racemic dl-isocitrate was 3.6 · 10−5 M and the apparent Km for NADP+ was 1.8 · 10−5 M. The enzyme was specific for the threo-d-isomer and required Mn2+ (5 · 10−4 M) for maximum activity. Partial activation also was achieved by Mg2+, Co2+ and Cd2+. The active species appears to exist as a polymeric unit consisting of monomers with a molecular weight of 15 000–20 000. The enzyme was sensitive to p-chloromercuribenzoate and inhibition was also obtained with ATP, pyrophosphate and by the concerted action of glyoxylate and oxaloacetate.

Purification and characterization of cytochrome O from Azotobacter vinelandii.

The membrane-bound cytochrome O has been solubilized from the Azotobacter vinelandii electron transport particle and further purified by use of conventional chromatographic procedures. The spectral characteristics as well as the other properties noted for purified cytochrome O are reported herein.

Enhanced activity of oligotrophic endogenous bacteria in clay‐rich sediments by nutrient injection

A controlled field experiment was performed in which the microbiology and geochemistry of clay‐rich fluvial‐deltaic sediments were characterized both before and after nutrient injection into a shallow well. Acetate addition (with nitrogen and phosphate) initially increased the heterotrophic bacteria population in the groundwater within 21 days after nutrient addition. Consumption of oxygen and injected nutrient resulted in an expected stimulation of copiotrophic bacterial growth (31–48 days), then a noticeable “trough phase”; reflecting minimal or no bacterial growth followed by a secondary peak reflecting another bacterial population growth surge (62–85 days). This secondary surge was apparently supported by the nutrients generated by the decomposing biomass (initial population peak), and by oxygen replenishment supplied by continual ground water flow. During the intervening trough phase, bacterial counts by most‐probable‐number analysis of soil samples indicated that the denitrifying population increase...

Non-pyridine nucleotide dependent l-(+)-glutamate oxidoreductase in Azotobacter vinelandii

Abstract l -(+)-Glutamate oxidation that is non-pyridine nucleotide dependent is readily carried out by a membrane-bound enzyme in Azotobacter vinelandii strain O. Enzyme activity concentrates in a membranous fraction that is associated with the Azotobacter electron transport system. This l -glutamate oxidation is not dependent on externally added NAD + , NADP + , FAD, or FMN for activity. O 2 , phenazine methosulfate and ferricyanide all served as relatively good electron acceptors for this reaction; while cytochrome c and nitrotetrazolium blue function poorly in this capacity. Paper chromatographic analyses revealed that the 2,4-dinitrophenylhydrazine derivative formed from the enzymatic oxidation of l -glutamate was α-ketoglutarate, while microdiffusion studies indicated that ammonia was also a key end product. These findings suggest that the overall reaction is an oxidative deamination. Ammonia formation was found to be stoichiometric with the amount of oxygen consumed (2 : 1 respectively, on a molar basis). The oxidation of glutamate was limited to the l -(+)-enantiomer indicating that this reaction is not the generalized type carried out by the l -amino acid oxidase. This oxidoreductase is functionally related to the Azotobacter electron transport system: (a) the activity concentrates almost exclusively in the electron transport fraction; (b) the l -glutamate oxidase activity is markedly sensitive to electron transport inhibitors, i.e. 2- n -heptyl-4-hydroxyquinoline- N -oxide, cyanide, and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione; and (c) spectral studies on the Azotobacter R 3 fraction revealed that a substantial amount of the flavoprotein (non-heme iron) and cytochrome ( a 2 , a 1 , b 1 , c 4 and c 5 ) are reduced by the addition of l -glutamate.

An Escherichia coli Mutant Conditionally Altered in Respiratory Chain Components

Nitrosoguanidine mutagenesis was employed to isolate an Escherichia coli mutant conditionally altered in respiratory chain components. Mutant R25 was able to grow on glucose, fructose, and glycerol but failed to grow on succinate and acetate (suc-). Also, R25 exhibited leaky growth on DL-lactate, fumarate, and malate (lct*). The lct* mutation pleiotropically affected a number of respiratory chain components and its expression was conditional with the growth substrate. Glucose-grown R25 resting cell suspensions oxidized DL-lactate and formate; however, these two substrates were not oxidized by fructose- or glycerol-grown cell suspensions. The same conditional pattern was observed for the concentration of cytochrome components, the membrane-associated oxidation of NADH and formate, and formate phenazine methosulfate (PMS) reductase activity; succinate oxidase and PMS reductase activities were not exhibited by membranes under any growth condition due to the suc- mutation. R25 membrane-associated H(+)-translocating ATPase activity was not conditional with the growth substrate. R25PC, a spontaneous lct+ suc- partial revertant of R25, did not exhibit the conditional pattern of R25. The lct* mutation was found to map in the 27-30-min region and the suc- mutation in the 15-17-min region of the E. coli genome. Two distinct classes of R25 P1kc transductants were isolated that differed in both their growth response on succinate and DL-lactate and their oxidase activities.

Studies on a non-pyridine nucleotide dependent, membrane-bound L-(+)-glutamate oxidoreductase in Azotobacter vinelandii.

Abstract A new type of membrane-bound oxidoreductase is described that carries out an oxidative deamination reaction that specifically involves L-glutamate. This enzyme is found in a subcellular fraction of Azotobacter vinelandii strain 0. It can oxidize L−(+)-glutamate using molecular oxygen and produces α-ketoglutarate and NH3 as end products. Neither NAD+ nor NADP+ are involved in this oxidation. The reaction is carried out by the membranous “R3” fraction which is obtained from sonically ruptured resting cells by differential centrifugation. In addition to O2, the electron acceptors that allowed for L-glutamate oxidation were phenazine methosulfate (PMS), K3Fe(CN)6, and 2, 6-dichloroindophenol (DCIP). This oxidation appears to be an integral part of the Azotobacter electron transport system as the L-glutamate oxidase rate is also highly sensitive to known electron transport inhibitors, i.e. , 2-n-hydroxy-4-quinoline-N-oxide, cyanide, and thenoyltrifluoroacetone. Spectral absorption studies on the Azotobacter R3 electron transport fraction revealed that the cytochrome and flavoprotein (non-heme iron) components also could be reduced completely upon the addition of L-glutamate. Preliminary results suggest that this is a new type of L-glutamate oxidoreductase that does not as yet have an Enzyme Commission number and appears to be (a) a specific flavoprotein enzyme that is not a type of L-amino acid oxidase, (b) tightly bound (and functionally attached) to the Azotobacter electron transport system, and (c) capable of carrying out specifically the oxidative deamination of L-glutamate in the absence of pyridine nucleotides.