Matthias Liebergesell

Formation of poly(3-hydroxyalkanoates) by phototrophic and chemolithotrophic bacteria

The formation of poly(3-hydroxyalkanoic acid), PHA, by various strains of chemolithotrophic and phototrophic bacteria has been examined. Chemolithotrophic bacteria were grown aerobically under nitrogen-limiting conditions on various aliphatic organic acids. Phototrophic bacteria were grown anaerobically in the light on a nitrogen-rich medium and were subsequently transferred to a nitrogen-free medium containing acetate, propionate, valerate, heptanoate or octanoate as carbon source. All 41 strains investigated in this study were able to synthesize and accumulate PHA. All 11 strains of chemolithotrophic bacteria and all 15 strains belonging to the non-sulfur purple bacteria synthesized a polymer, which contained 3-hydroxy-valerate (3HV) beside 3-hydroxybutyrate (3HB), if the cells were cultivated in the presence of propionate, valerate or heptanoate. Many non-sulfur purple bacteria synthesized copolyesters of 3HB and 3HV even with acetate as carbon source. In contrast, most sulfur purple bacteria did not incorporate 3HV at all. Among 15 strains tested, only Chromatium vinosum strain 1611, C. purpuratum strain BN5500 and Lamprocystis roseopersicina strain 3112 were able to synthesize polyesters containing 3HV with propionate, valerate or heptanoate as carbon source.

Anaylsis of polyhydroxyalkanoic acid-biosynthesis genes of anoxygenic phototrophic bacteria reveals synthesis of a polyester exhibiting an unusal composition

From genomic libraries of purple sulphur bacteria, fragments were cloned that encoded for proteins involved in the synthesis of poly(3-hydroxyalkanoic acids), PHA. A 12.5- and a 15.0- plus a 15.6-kbp EcoRI-restriction fragment of Ectothiordospira shaposhnikovii of Thiocapsa pfennigii, respectively, which hybridized with a fragment encoding the Alcaligenenes eutrophus PHA-biosynthesis operon, were identified in δL47 libraries, whereas an 18.0-kbp EcoRI fragment of Lamprocytis roseopersicina, which phenotypically complemented a PHA-neagative mutant of A. eutrophus, was identified in a pVK100 cosmid library. Hybridization studies and enzymatic analysis of crued extracts derived from transconjugants of Escherichia coli and A. eutrophus harbouring these fragments revealed the presence of the genes for NADH-dependent acetoacetyl-CoA reductase and/or PHA synthase. The PHA-biosynthesis genes of T. pfennigii and L. roseopersicina as wells as of Chromatium vinosum, Thiocystis violacea, Rhodospirillum rubrum and Rodobacter sphaeroides were then analysed for thire ability to confer synthesis of PHA other poly(3-hydroxybutric acid) to PHA-negative mutants of PHA-accumulating bacteria. The most striking result was that a fragment harbouring the PHA-synthase gene of T. pfennigii conferred the ability to synthesize a polymer consisting of almost equimolar amounts of 3-hydroxybutyrate (48.5 mol%) and 3-hydroxyhexanote (47.3%) plus a small amount of 3-hydroxyoctanoate (4.2 mol %) to a PHA-negative mutant of Pseudomonas putida. A niosynthetic polyester with this composition has not been described before.

Cloning and molecular analysis of the poly(3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea

From a genomic library of Thiocystis violaceae strain 2311 in λL47, two adjacent EcoRI restriction fragments of 5361 base pairs (bp) and of 1978 bp were cloned. The 5361-bp EcoRI restriction fragment hybridized with a DNA fragment harbouring the Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthase operon (phbCAB) and restored the ability to synthesize and accumulate PHA in PHA-negative mutants derived from A. eutrophus. The nucleotide sequence analysis of both fragments revealed five open-reading frames (ORFs); at least three of them are probably relevant for PHA biosynthesis. The amino acid sequences of the putative proteins deduced from these genes indicate that they encode a β-ketothiolase [phbATv, relative molecular mass (Mr) 40850], which exhibited 87.3% amino acid identify with the β-ketothiolase from Chromatium vinosum. The amino acid sequences of the putative proteins deduced from ORF2Tv (Mr 41 450) and phbCTv (Mr 39 550), which were located upstream of and antilinear to phbATv, exhibited 74.7% and 87.6% amino acid identify, respectively, with the corresponding gene products of C. vinosum. Downstream of and antilinear to phbCTv was located ORF5, which encodes for a protein of high relative molecular mass (Mr 76428) of unknown function. With respect to the divergent organisation of ORF2Tv and phbCTv on one side and of phbATv on the other side and from the homologies of the putative gene products, this region of the T. violaceae genome resembled very much the corresponding region of C. vinosum, which was identified recently.

Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme

Abstract The PHA synthase structural gene of Thiocapsa pfennigii was identified and subcloned on a 2.8-kbp BamHI restriction fragment, which was cloned recently from a genomic 15.6-kbp EcoRI restriction fragment. Nucleotide sequence analysis of this fragment revealed three open reading frames (ORFs), representing coding regions. Two ORFs encoded for the PhaE (Mr 40,950) and PhaC (Mr 40,190) subunits of the PHA synthase from T. pfennigii and exhibited high homology with the corresponding proteins of the Chromatium vinosum (52.8% and 85.2% amino acid identity) and the Thiocystis violacea (52.5% and 82.4%) PHA synthases, respectively. This confirmed that the T. pfennigii PHA synthase was composed of two different subunits. Also, with respect to the molecular organization of phaE and phaC, this region of the T. pfennigii genome resembled very much the corresponding regions of C. vinosum and of Thiocystis violacea. A recombinant strain of Pseudomonas putida, which overexpressed phaE and phaC from T. pfennigii, was used to isolate the PHA synthase by a two-step procedure including chromatography on Procion Blue H-ERD and hydroxyapatite. The isolated PHA synthase consisted of two proteins exhibiting the molecular weights predicted for PhaE and PhaC. Hybrid PHA synthases composed of PhaE from T. pfennigii and PhaC from C. vinosum and vice versa were constructed and functionally expressed in a PHA-negative mutant of P. putida; and the resulting PHAs were analyzed.

THE PHOTOPHOBIC RESPONSE OF VARIOUS SULFUR AND NONSULFUR PURPLE BACTERIA

Abstract— The photophobic response of 34 strains of nonsulfur and sulfur purple bacteria was examined with respect to response‐eliciting light intensities. The bacteria were grown in defined synthetic media or in Winogradsky columns. Two population methods based on Engelmanns light trap were used to determine the discrimination thresholds of the bacteria. A single‐side irradiation method allowed the estimation of approximate values, while the double‐side irradiation method provided more exact values of the discrimination threshold. Sixteen strains belonging to 9 different species exhibited discrimination thresholds between 0.7% and 2.6%. The motility of the other 18 strains proved to be insufficient to measure light sensitivities with the methods used. The effect of various environmental factors on the light sensitivies of Chromatium vinosum D and Rhodospirillum rubrum Ha was examined. The measurements and observations made in this work recommend strains of Rhodospirillum rubrum and Chromatium vinosum as model organisms for further studies.

New knowledge about the PHA-locus and P(3HB) granule-associated proteins in Chromatium vinosum

SummaryThe isolation of poly(3-hydroxybutyric acid) granules of Chromatium vinosum D was re-examined. Beside the PHA synthase and a 17 kDa protein, a 14 kDa protein was identified as predominant granule-associated protein. The Mr as well as the N-terminal amino acid sequence exhibited identity to ORF5Cv, which is located within the pha-locus of C. vinosum. In addition, sequence alignements revealed new information about ORF4Cv, which is also located within the pha-locus, and about the 17 kDa protein, which exhibited homology to heat shock proteins recently detected in Escherichia coli.

Molecular analysis of the Aureobasidium pullulans ura3 gene encoding orotidine-5'-phosphate decarboxylase and isolation of mutants defective in this gene.

Abstract Orotidine-5′-phosphate decarboxylase (OMP decarboxylase) catalyses the final step in the pyrimidine biosynthesis, the conversion of orotidine-5′-phosphate (OMP) to uridine-5′-phosphate. The ura3 gene of Aureobasidium pullulans, encoding OMP decarboxylase, was isolated from an Aureobasidium genomic library constructed in the plasmid pBlueskriptSK−. The ura3 gene of A. pullulans has an open reading frame of 271 amino acid residues. Analysis of the sequence revealed the presence of two introns. In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Aspergillus niger, Neurospora crassa, Phycomyces blakesleeanus and Homo sapiens. The ura3 gene is the third Aureobasidium gene that has been cloned and analysed. We have also isolated ura3 mutants by selection of ethyl methanesulphonate mutagenised cells on 5-fluoroorotic acid. Transformation of these A. pullulans mutant strains to prototrophy showed the functionality of the cloned gene.