Accumulation of Poly (β-Hydroxybutyrate) by Halobacteria

Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas fragi, Escherichia coli, and Salmonella "anatum" were challenged with butylated hydroxyanisole (BHA). Susceptibility was measured as the concentration of BHA required to cause a 90% reduction in bacterial survivors. Staphylococcus aureus LP and P. fragi were two of the most resistant species examined; C. perfringens and P. fluorescens were the most susceptible. Gram stain reaction was found not to be a strict indicator of bacterial susceptibility to BHA. There was no obvious relationship between individual fatty acids and susceptibility. The ratio of saturated to unsaturated fatty acids in the total lipid fraction of only the gram-positive species was related to susceptibility. The ratios of saturated to unsaturated fatty acids of other fractions were not related to susceptibility.     

Involvement of the Reserve Material Poly-β-Hydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization

When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.     

The ntrB and ntrC Genes Are Involved in the Regulation of Poly-3-Hydroxybutyrate Biosynthesis by Ammonia in Azospirillum brasilense Sp7

Azospirillum brasilense Sp7 and its ntrA (rpoN), ntrBC, and ntrC mutants have been evaluated for their capabilities of poly-3-hydroxybutyrate (PHB) accumulation in media with high and low ammonia concentrations. It was observed that the ntrBC and ntrC mutants can produce PHB in both low- and high-C/N-ratio media, while no significant PHB production was observed for the wild type or the ntrA mutant in low-C/N-ratio media. Further investigation by fermentation analysis indicated that the ntrBC and ntrC mutants were able to grow and accumulate PHB simultaneously in the presence of a high concentration of ammonia in the medium, while little PHB was produced in the wild type and ntrA (rpoN) mutant during active growth phase. These results provide the first genetic evidence that the ntrB and ntrC genes are involved in the regulation of PHB synthesis by ammonia in A. brasilense Sp7.     

Floc Formation by Azospirillum lipoferum Grown on Poly-β-Hydroxybutyrate

Azospirillum lipoferum RG6xx was grown under conditions similar to those resulting in encystment of Azotobacter spp. A. lipoferum produced cells of uniform shape when grown on nitrogen-free β-hydroxybutyrate agar. Cells accumulated poly-β-hydroxybutyrate and often grew as chains or filaments that eventually lost motility and formed capsules. Within 1 week, vegetative A. lipoferum inocula were converted into microflocs arising from filaments or chains. Cells within microflocs were pleomorphic, contained much poly-β-hydroxybutyrate, and were encapsulated. Some cells had a cystlike morphology. Up to 57% of the dry weight of encapsulated flocs was poly-β-hydroxybutyrate, whereas vegetative cells grown in broth with combined nitrogen had only 3% of their dry weight as poly-β-hydroxybutyrate. Neither encapsulated cells in flocs nor nonencapsulated vegetative cells were significantly desiccation resistant. Under starvation conditions (9 days) only 25% of encapsulated cells remained viable, whereas vegetative cells multiplied severalfold. In short-term germination experiments with encapsulated flocs, nitrate, ammonium, and soil extract promoted formation of motile vegetative cells. Most cells in treatments lacking combined nitrogen eventually depleted their visible poly-β-hydroxybutyrate reserves without germinating. The remaining cells retained the reserve polymer and underwent size reduction.     

Identification and isolation of genes involved in poly(beta-hydroxybutyrate) biosynthesis in Azospirillum brasilense and characterization of a phbC mutant

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly(beta-hydroxybutyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments, thus showing great potential for the improvement of bacterial inoculants for plants and soils. The three genes that are considered to be essential in the PHB biosynthetic pathway, phbA (beta-ketothiolase), phbB (acetoacetyl coenzyme A reductase), and phbC (PHB synthase), were identified in Azospirillum brasilense strain Sp7, cloned, and sequenced. The phbA, -B, and -C genes were found to be linked together and located on the chromosome. An A. brasilense phbC mutant was obtained by insertion of a kanamycin resistance cassette within the phbC gene. No PHB production was detected in this mutant. The capability of the wild-type strain to endure starvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, root adhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in the phbC mutant strain than in the wild type.     

Isolation and Properties of a β-Alanine Transaminaseless Mutant of Pseudomonas fluorescens

beta-Alanine catabolism in Pseudomonas fluorescens is initiated by the enzyme beta-alanine transaminase. We have isolated mutants which fail to produce this enzyme and thus cannot grow on beta-alanine as the sole nitrogen source. The accumulation of beta-alanine-1-(14)C has been studied in one of these mutants, strain 67, and in the wild type. In the mutant, beta-alanine remains in a stable intracellular pool, whereas in the wild type conversion of beta-alanine to an intermediate, presumably malonate semialdehyde, and to CO(2) can be detected. The membrane transport system for beta-alanine can be conveniently studied in this transaminaseless mutant.     

Isolation and Characterization of a Bacillus cereus Mutant Strain Hyperproductive of Exo-β-Amylase

Starting with a strain of Bacillus cereus excreting about 40-fold more β-amylase than does the original wild-type strain, we isolated, after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine, a strain designated BQ10-S1 SpoIII which showed under optimal conditions a further 5.5-fold increase in β-amylase activity. The amylase production of this strain was observed to increase in the presence of 0.5% glucose or 1% maltose and, more markedly, in the presence of 2% soluble starch in the culture medium. The enzyme produced by this strain was immunologically identical to the wild-type enzyme, suggesting that either the copy number of the gene or the efficiency of enzyme synthesis from it, or both, are altered in this strain.     

Metabolism of Poly-β-Hydroxybutyrate: Effect of Mild Alkaline Extraction on Native Poly-β-Hydroxybutyrate Granules

Mild alkaline extraction of native poly-beta-hydroxybutyrate (PHB) granules results in the solubilization of a protein fraction. Both the solubilized protein fraction and the extracted granules are essentially devoid of PHB synthetase activity unless recombined. The protein fraction has been separated by chromatography into two components (A-I and A-II). A-I but not A-II can be recombined with extracted granules to give rise to PHB synthetase activity. Extracted granules no longer require pretreatment with activator or trypsin but are directly susceptible to hydrolysis by Rhodospirillum rubrum depolymerase. Addition of A-II or A-I prevents the direct hydrolysis by depolymerase. The inhibition is reversed by activator or trypsin. We conclude that native granules are associated with a protein inhibitor which prevents the hydrolysis of PHB by depolymerase unless the protein is destroyed by trypsin, removed by alkaline extraction, or modified by activator.     

Quantifying Nonspecific TEM β-Lactamase (blaTEM) Genes in a Wastewater Stream

To control the antibiotic resistance epidemic, it is necessary to understand the distribution of genetic material encoding antibiotic resistance in the environment and how anthropogenic inputs, such as wastewater, affect this distribution. Approximately two-thirds of antibiotics administered to humans are β-lactams, for which the predominant bacterial resistance mechanism is hydrolysis by β-lactamases. Of the β-lactamases, the TEM family is of overriding significance with regard to diversity, prevalence, and distribution. This paper describes the design of DNA probes universal for all known TEM β-lactamase genes and the application of a quantitative PCR assay (also known as Taqman) to quantify these genes in environmental samples. The primer set was used to study whether sewage, both treated and untreated, contributes to the spread of these genes in receiving waters. It was found that while modern sewage treatment technologies reduce the concentrations of these antibiotic resistance genes, the ratio of bla_TEM genes to 16S rRNA genes increases with treatment, suggesting that bacteria harboring bla_TEM are more likely to survive the treatment process. Thus, β-lactamase genes are being introduced into the environment in significantly higher concentrations than occur naturally, creating reservoirs of increased resistance potential.     

β-Galactosidase from Termination and Deletion Mutant Strains

β-Galactosidase fragments were isolated from strains of Escherichia coli with mutations in the lacZ gene. The polypeptide obtained from a termination mutant (lacZNG125) appeared to be the intact gene product, containing the first half of the β-galactosidase amino acid sequence. From an internal deletion mutant strain (lacZU163), an aggregate was obtained of several partially degraded polypeptides. Each of these was smaller than predicted from genetic data for the fragment. Introduction of the lacZU163 mutation into a protein degradation-deficient strain (Deg⁻) resulted in the protection of the amino-terminal region of the protein. Some of the BrCN peptides from the U163 polypeptides were separated and identified. From such experiments it was shown that in both Deg⁻ and Deg⁺ strains the COOH-terminal region is rapidly degraded. This indicates that the complete gene product of lacZU163 has not been detected. The use of genetically defined enzyme fragments in studying structure-function relationships and in determination of primary structure is discussed.     

Involvement of glnB, glnZ, and glnD Genes in the Regulation of Poly-3-Hydroxybutyrate Biosynthesis by Ammonia in Azospirillum brasilense Sp7

The role of three key nitrogen regulatory genes, glnB (encoding the P_II protein), glnZ (encoding the P_z protein), and glnD (encoding the GlnD protein), in regulation of poly-3-hydroxybutyrate (PHB) biosynthesis by ammonia in Azospirillum brasilense Sp7 was investigated. It was observed that glnB glnZ and glnD mutants produce substantially higher amounts of PHB than the wild type produces during the active growth phase. glnB and glnZ mutants have PHB production phenotypes similar to that of the wild type. Our results indicate that the P_II-P_z system is apparently involved in nitrogen-dependent regulation of PHB biosynthesis in A. brasilense Sp7.     

Identification of Novel Temperature-sensitive Lethal Alleles in Essential β-Tubulin and Nonessential α2-Tubulin Genes as Fission Yeast Polarity Mutants

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1–15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two α-tubulin genes) and alp12 to nda3 (the single β-tubulin gene). atb2⁺ is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between α/β-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2⁺; overexpression of atb2⁺ lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.     

Identification and Characterization of Pancreatic Eukaryotic Initiation Factor 2 α-Subunit Kinase, PEK, Involved in Translational Control

In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). In mammals, the phosphorylation was shown to be carried out by eIF-2α kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2α kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2α kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced in Escherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2α on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In the Saccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2α kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2α. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2α kinase plays an important role in translational control from nematodes to mammals.     

Determination of Poly-β-Hydroxybutyrate and Poly-β-Hydroxyvalerate in Activated Sludge by Gas-Liquid Chromatography

A convenient gas-liquid chromatography procedure to quantify poly-β-hydroxybutyrate and poly-β-hydroxyvalerate in activated sludge was developed by combining lyophilization of the samples, purification of the chloroform phase by water reextraction, and the use of capillary columns. With a flame ionization detector the sensitivity was estimated at 10⁻⁵ g/liter.     

Encystment and Polymer Production by Azotobacter vinelandii in the Presence of β-Hydroxybutyrate

Cells of Azotobacter vinelandii encysted in Burk's nitrogen-free liquid media which had been supplemented with n-butyl alcohol, β-hydroxybutyrate, or crotonate. Butyraldehyde and butyrate did not influence the extent of encystment. In the absence of glucose, β-hydroxybutyrate enhanced the rate and extent of encystment. In the presence of glucose, it promoted abortive encystment, which was manifested by the disorganization of the exine and the release of a highly viscous material into the medium. The soluble, viscous polymer was separated from the medium by a series of ethyl alcohol precipitations and identified as a mucopeptide. It was cleaved by treatment with lysozyme and lysostaphin with a concomitant increase in reducing power. It contained 13.9% N; 56% amino acids, as alanine (alanine, lysine, and glutamic acids); and 42% hexosamines. The polymer appeared to be similar to a noncross-linked peptidoglycan.