A bit of history.

Reviews of scientific literature began to appear in the 17th century. Journals dedicated to them soon followed, leading eventually to this one, which emerged in the 1930s as Bacteriological Reviews; it adapted to the many changes in our fluid discipline, evolving into the present, much broader Microbiology and Molecular Biology Reviews.

Molecular characterization of nosA, a Pseudomonas stutzeri gene encoding an outer membrane protein required to make copper-containing N2O reductase.

A Pseudomonas stutzeri gene (nosA) encoding an outer membrane protein was cloned into the broad-host-range vector pRK290 and expressed in a mutant lacking the protein. Deletion analysis identified the approximate extent of the nosA region which was sequenced, and it was found to contain an open reading frame encoding 683 amino acids including a presumed signal sequence of 44 amino acids. The putative processed form had a molecular weight of 70,218, characteristics typical of outer membrane proteins, and considerable amino acid sequence homology with Escherichia coli BtuB. A short stretch of amino acids was homologous with the E. coli TonB-dependent outer membrane proteins, BtuB, IutA, FepA, and FhuA, suggesting a homologous function: interaction with a periplasmic protein or uptake of a specific substrate.

Effect of growth temperature on folding of carbamoylphosphate synthetases of Salmonella typhimurium and a cold-sensitive derivative.

The properties of homogeneous preparations of carbamoylphosphate synthetase (CPSase) from wild-type Salmonella typhimurium and a cold-sensitive derivative grown at different growth temperatures were examined. For the cold-sensitive mutant, the affinity for glutamine of the form of CPSase synthesized at 20 degrees C was lower than that of the form of the enzyme synthesized at 37 degrees C, regardless of the assay temperature. Thus, the cold sensitivity of the mutant reflects an effect of temperature on the synthesis of the enzyme rather than the activity of the folded enzyme. The two forms also differed in sensitivities to polyclonal antibodies as well as denaturational enthalpies. The combined results support the hypothesis that carAB mutations conferring cold sensitivity identify amino acid residues that are critical in the folding of CPSase. Quite unexpectedly, certain kinetic properties of cloned parent CPSase were also dependent on the growth temperature, although to a much lesser extent than those of the cold-sensitive mutant. The specific activity of wild-type CPSase synthesized at 15 degrees C was 60% of that synthesized at 37 degrees C. Further, CPSase synthesized at 15 degrees C was less thermostable than the enzyme synthesized at 37 degrees C; the difference in stability (delta G) is estimated to be 4,500 cal mol-1. Thus, variation of temperature within the physiological range for growth influences the folding and consequently the properties of CPSase from wild-type S. typhimurium.

A NosA-specific bacteriophage can be used to select denitrification-defective mutants of Pseudomonas stutzeri.

phi PS5, a double-stranded DNA bacteriophage of Pseudomonas stutzeri JM604 that adsorbs specifically to the outer-membrane protein NosA, was isolated from stagnant irrigation ditch water. Mutant strains that do not produce NosA are resistant to phi PS5 and cannot grow anaerobically with N2O as the sole electron acceptor. phi PS5 did not adsorb to nosA mutants and adsorption to the wild-type strain was reduced when cells were grown with a high concentration of copper, a condition that represses the synthesis of NosA. The isolation of spontaneous phi PS5-resistant mutants yielded strains that were clearly defective in growth on N2O at about a 10% incidence. About half of these strains could respire N2O when supplied with a high concentration of exogenous copper.

Properties of a Pseudomonas stutzeri outer membrane channel-forming protein (NosA) required for production of copper-containing N2O reductase.

A protein (NosA) in the outer membrane of Pseudomonas stutzeri that is required for copper to be inserted into N2O reductase has been extracted and purified to homogeneity. The purified protein could form channels in black lipid bilayers. Like N2O reductase, NosA contained copper and was only made anaerobically. In contrast to N2O reductase, its synthesis was repressed by exogenous copper (but not by Mn, Co, Ni, Zn, or Fe). Also in contrast to N2O reductase, NosA homologs were not immunologically detectable in Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, or other strains of P. stutzeri.

A Pseudomonas stutzeri outer membrane protein inserts copper into N2O reductase.

Among a set of frameshift mutagen (ICR-191; Polysciences, Inc.)-induced mutations that confer inability to grow anaerobically with N2O as the sole electron acceptor, one class was found that produced an inactive N2O reductase which lacked copper. All of these mutant strains failed to produce a 61,000-Mr protein located in the outer membrane. This protein, termed NosA, seems not to be responsible for bringing copper into the cell because the mutant strains and their parent were similarly sensitive to the copper content of the growth medium and no intermediate copper concentration in the medium permitted the mutant strains (nosA) to grow anaerobically with N2O as the sole electron acceptor. We conclude that NosA is necessary to insert copper into N2O reductase or to maintain it there.

Thymidine salvage in Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous expression of Escherichia coli thymidine kinase gene.

Unlike enteric bacteria, Pseudomonas spp. generally lack thymidine phosphorylase and thymidine kinase activities, thus preventing their utilization of exogenous thymine or thymidine and precluding specific radioactive labeling of their DNA in vivo. To overcome this limitation, a DNA fragment encoding thymidine kinase (EC 2.7.1.21) from Escherichia coli was cloned into pKT230, a small, broad-host-range plasmid derived from plasmid RSF1010. From transformed E. coli colonies, the recombinant plasmid bearing the thymidine kinase gene was conjugally transferred to Pseudomonas stutzeri, Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes. Thymidine kinase activity was expressed in all of these species, and all gained the ability to incorporate exogenous [2-14C]thymidine into their DNA. Thymidine incorporation into P. stutzeri was enhanced 12-fold more in mutants lacking thymidylate synthetase activity. These mutants produced higher levels of thymidine kinase and were thymidine auxotrophs; thymineless death resulted from removal of thymidine from a growing culture.

Isolation and characterization of mutant Pseudomonas aeruginosa strains unable to assimilate nitrate.

Single-site mutants of Pseudomonas aeruginosa that lack the ability aerobically to assimilate nitrate and nitrite as sole sources of nitrogen have been isolated. Twenty-one of these have been subdivided into four groups by transductional analysis. Mutants in only one group, designated nis, lost assimilatory nitrite reductase activity. Mutants in the other three transductional groups, designated ntmA, ntmB, ntmC, display a pleiotropic phenotype: utilization of a number of nitrogen-containing compounds including nitrite as sole nitrogen sources is impaired. Assimilatory nitrite reductase was shown to be the major route by which hydroxylamine is reduced in aerobically-grown cells.

Chromosomal location and function of genes affecting Pseudomonas aeruginosa nitrate assimilation.

Seven known genes control Pseudomonas aeruginosa nitrate assimilation. Three of the genes, designated nas, are required for the synthesis of assimilatory nitrate reductase: nasC encodes a structural component of the enzyme; nasA and nasB encode products that participate in the biosynthesis of the molybdenum cofactor of the enzyme. A fourth gene (nis) is required for the synthesis of assimilatory nitrite reductase. The remaining three genes (ntmA, ntmB, and ntmC) control the assimilation of a number of nitrogen sources. The nas genes and two ntm genes have been located on the chromosome and are well separated from the known nar genes which encode synthesis of dissimilatory nitrate reductase. Our data support the previous conclusion that P. aeruginosa has two distinct nitrate reductase systems, one for the assimilation of nitrate and one for its dissimilation.

Evidence for an active role of donor cells in natural transformation of Pseudomonas stutzeri.

The transfer of chromosomal genes in a cell mat of Pseudomonas stutzeri was ca. 10(3) times more efficient per microgram of DNA if DNA was added as a constituent of intact donor cells rather than as a solution. Such intact cell-mediated transfer appears to depend on cell contact. It is independent of the presence of plasmids in donor strains and is DNase I sensitive, thus fitting the usual definition of transformation. It is bidirectional: cells of either strain in a transformation mixture served as the donor and recipients. The donor function in cell contact transformation was inhibited by nalidixic acid but was unaffected by rifampin and streptomycin at growth-inhibiting concentrations. Concentrations of nalidixic acid sufficient to inhibit donor function completely had no effect on the ability of nalidixic acid-resistant recipients to take up DNA from solution. These experiments suggest that certain cells donate DNA to others in the cell mat: they argue against the hypothesis that the function of donor cells is merely cell lysis.

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans.

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.

Location on the Salmonella typhimurium chromosome of the gene encoding nucleoside diphosphokinase (ndk).

The gene encoding nucleosidediphosphate kinase (ndk) was located at 55 units on the Salmonella typhimurium chromosome. The ndk locus was 83% cotransducible with hisS and 2% cotransducible with glyA in phage P22-mediated crosses. A nucleosidediphosphate kinase mutant that produced only 10% of the wild-type enzyme activity (ndk-1) grew normally and produced a heat-labile enzyme.

Pseudomonas stutzeri and related species undergo natural transformation.

Cells of Pseudomonas stutzeri are naturally transformed by homologous chromosomal DNA; they do not require chemical treatment to become competent. This capacity to undergo natural transformation was found to be shared by the closely related species P. mendocina, P. alcaligenes, and P. pseudoalcaligenes, but was not detectable in strains of P. aeruginosa, P. perfectomarinus, P. putida, P. fluorescens, or P. syringae. P. stutzeri could be transformed either on plates or in liquid medium. Only double-stranded chromosomal DNA was effective; single-stranded DNA and plasmid DNA were not. DNA fragments larger than 10 kilobase pairs were more effective than smaller fragments. The transformation frequency was proportional to DNA concentration from 1 ng/ml to 1 microgram/ml; higher concentrations were saturating. The maximum frequency, about 10(-4) transformants per recipient cell, was obtained with cells from a culture in the early stationary growth phase. A variety of chromosomal mutations have been transformed, including mutations to auxotrophy and to antibiotic resistance. Other systems for genetic exchange in P. stutzeri have not yet been found; transformation offers a means for the genetic analysis of this metabolically versatile organism.

Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa.

Dissimilatory nitrate reductase was purified to homogeneity from anaerobic cultures of the denitrifying bacterium Pseudomonas aeruginosa. The following procedures were used in the rapid isolation of this unstable enzyme: induction by nitrate in semianaerobic cell suspension, heat-stimulated activation and solubilization from the membrane fraction, and purification by hydrophobic interaction chromatography. The molecular weight of the purified enzyme was estimated by nondenaturing polyacrylamide gel electrophoresis, sucrose density gradient sedimentation, and gel filtration chromatography. Subunit molecular weights were estimated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The active enzyme monomer, with a molecular weight of 176,000 to 260,000 (depending upon the method of determination), was composed of subunits with molecular weights of approximately 64,000 and 118,000. The monomer aggregated to form an inactive tetramer of about 800,000 molecular weight. Purified enzyme exhibited a broad pH optimum, between 6.5 and 7.5. Kinetic studies showed that the apparent Km was 0.30 mM for nitrate, and 2.2 to 2.9 microM for dithionite-reduced benzyl viologen. Azide was an effective inhibitor: the concentration required for half-maximal inhibition was 21 to 24 microM. Azide inhibition was competitive with nitrate (Ki = 2.0 microM) but uncompetitive with reduced benzyl viologen (Ki = 25 microM). Based upon spectral evidence, the purified molybdo-enzyme had no associated cytochromes but did contain nonhaem iron that responded to dithionite reduction and nitrate oxidation. The enzyme that was purified after being heat solubilized from membranes had properties essentially identical to those of the enzyme that was purified after deoxycholate solubilization.

A regulatory gene (use) affecting the expression of pyrA and certain other pyrimidine genes.

The use-1 mutation in Salmonella typhimurium confers a complex and pleiotrophic phenotype which is primarily characterized as a temperature-dependent sensitivity to uracil. This sensitivity can be reversed by arginine or citrulline, but not by ornithine, suggesting that the use-1 mutation affects the synthesis or the activity (or both) of carbamoylphosphate synthetase or ornithine carbamoyltransferase (or both). Activity measurements showed that use-1 caused superrepression of both of these enzymes, especially when uracil was present in the medium. Dihydro-orotase and dihydro-orotate oxidase were also superrepressed, but aspartate carbamoyltransferase and orotate phosphoribosyltransferase were not. Lowered nucleotide triphosphate and guanosine tetra- and pentaphosphate pools in use-1 strains indicated that the mutation affected synthesis or breakdown of all of these phosphorylated compounds, but the UTP pool increased by a larger relative factor in use-1 strains in the presence of uracil. The uracil-sensitive phenotype of the use-1 mutation is a complex response to several environmental factors: temperature, aerobiosis, carbon sources, and uracil concentration. Uracil sensitivity was eliminated by alteration of one or more of these factors. Uracil sensitivity was suppressed by several genetic alterations. These include introduction into use-1 strains of a multi-copy ColE1 derivative which carries the structural gene(s) for carbamoylphosphate synthetase, episomes that carry use, mutations including argR and pyrH, and various unclassified intergenic suppressor mutations. These genetic changes increased significantly the expression of carbamoylphosphate synthetase or ornithine carbamoyltransferase (or both). The activity of use-1 is not known, but the facts that it altered expression of at least four unlinked genes (pyrA, pyrC, pyrD, and argI) and that the Escherichia coli F'133 complemented it establish it as a trans-acting regulatory factor.

Isolation and mapping of a uracil-sensitive mutant of Salmonella typhimurium.

A uracil-sensitive mutant of Salmonella typhimurium was isolated by diethyl sulfate mutagenesis and penicillin counterselection. This mutation identifies a new Salmonella gene that is well separated from the structural genes for arginine and pyrimidine biosynthesis. The use-1 mutation was located between the ilv gene cluster (isoleucine-valine operon) and hisR (structural gene for histidine tRNA) at 83 map units.

Infection of Salmonella typhimurium with coliphage Mu d1 (Apr lac): construction of pyr::lac gene fusions.

A procedure was developed for introducing the coliphage Mu d1 (Apr lac) into Salmonella typhimurium in order to construct gene fusions that place the structural genes of the lac operon under the control of the promoter-regulatory region of other genes. To introduce Mu d1 from Escherichia coli K-12 into S. typhimurium, which is normally not a host for Mu, we first constructed an E. coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid helper phage (MuhP1) that confers the P1 host range. A lysate prepared from this strain was used to infect a P1-sensitive (i.e., galE), restriction-deficient, modification-proficient strain of S. typhimurium, and a double lysogen carrying Mu d1 and MuhP1 was isolated. Induction of the latter strain produced lysates capable of infecting and generating gene fusions in P1-sensitive strains of S. typhimurium. In this paper we describe the construction of pyr::lac fusions by this technique.