ALG1-CDG: a new case with early fatal outcome.

Congenital disorders of glycosylation (CDG) are a growing group of inherited metabolic disorders where enzymatic defects in the formation or processing of glycolipids and/or glycoproteins lead to variety of different diseases. The deficiency of GDP-Man:GlcNAc2-PP-dolichol mannosyltransferase, encoded by the human ortholog of ALG1 from yeast, is known as ALG1-CDG (CDG-Ik). The phenotypical, molecular and biochemical analysis of a severely affected ALG1-CDG patient is the focus of this paper. The patient's main symptoms were feeding problems and diarrhea, profound hypoproteinemia with massive ascites, muscular hypertonia, seizures refractory to treatment, recurrent episodes of apnoea, cardiac and hepatic involvement and coagulation anomalies. Compound heterozygosity for the mutations c.1145T>C (M382T) and c.1312C>T (R438W) was detected in the patient's ALG1-coding sequence. In contrast to a previously reported speculation on R438W we confirmed both mutations as disease-causing in ALG1-CDG.

Structural and functional characterization of the Bacillus megaterium uvrBA locus and generation of UV-sensitive mutants.

The Bacillus megaterium genes uvrB and uvrA, encoding two subunits of the (A)BC excinuclease, which is responsible for nucleotide excision repair, were isolated and functionally characterized. RNA analyses revealed co-transcription of both genes probably forming a bicistronic operon. Expression of uvrB and uvrA was inducible by the DNA-damaging agent mitomycin C. This finding agrees with the presence of a potential DinR box within the uvrBA promoter. Single inactivation of uvrB or uvrA as well as the parallel knockout of both genes resulted in mutants highly sensitive to UV irradiation. Thus, this locus represents an attractive target for generating biologically safe containment strains of B. megaterium.

Isolation of phages infecting Actinoplanes SN223 and characterization of two of these viruses.

Phages infecting the industrially important Actinoplanes strain SN223 were isolated from soil samples collected at the shores of inland waters in Germany. The genome sizes range from 53 kb to 58 kb. Preliminary analyses revealed G+C contents comparable with the G/C bias of the host. Electron microscopy of three selected viruses displayed no obvious morphological differences, the phage heads being icosahedral and their tails non-contractible. Two of the phages (phiAsp2, phiAsp3.1) characterized in more detail are capable of provoking putative pseudolysogenic growth of the host bacterium. The carrier state for phiAsp2, in which cells are tightly packed with viruses, was demonstrated by electron microscopy. The latter phage is apparently widely distributed, as it was isolated from regions which are distantly located, i.e. more than 600 km apart from each other.

Identification and functional characterization of a type I signal peptidase gene of Bacillus megaterium DSM319.

The sipM gene of Bacillus megaterium encoding a type I signal peptidase (SPase) was isolated and structurally characterized. RNA analysis revealed a transcript size in accordance with a bicistronic operon comprising sipM and an adjacent open reading frame. Inactivation of sipM by targeted gene disruption could not be achieved, indicating its essential role for cell viability since there might be no other type I SPase of major importance present in B. megaterium. Plasmid-assisted amplification of the gene resulted in an increase in activity of the heterologous glucanase used as an extracellular reporter, suggesting a potential bottleneck for protein secretion within this species.

Structural and functional analysis of the killer element pPin1-3 from Pichia inositovora.

Strains of the yeast Pichia inositovora that carry the linear plasmids pPin1-1 (18 kb) and pPin1-3 (10 kb) display a killer activity towards Saccharomyces cerevisiae. Cloning and sequencing of the smaller plasmid, pPin1-3, revealed that it is 9683 bp long and has 154-bp terminal inverted repeats. Comparison of pPin1-3 with the only other completely sequenced killer plasmid, pGKL1 of Kluyveromyces lactis, revealed differences in genome organization. The Pichia element has four ORFs that account for 95% of the sequence. ORF1 is homologous to the putative immunity gene of the K. lactis system. A viral B-type DNA polymerase is encoded by ORF2. The predicted product of ORF3 displays similarities to the alpha- and beta-subunits of the heterotrimeric K. lactis killer toxin, also known as zymocin. A cysteine-rich chitin-binding site and a chitinase signature, characteristic for the alpha-subunit of zymocin were identified in Orf3p. Chitin affinity chromatography and Western analysis confirmed the plasmid specific expression and secretion of a protein that cross-reacts with an antibody raised against the alpha-subunit of K. lactis zymocin. Disruption of the major chitin synthase-gene ( CHS3) renders S. cerevisiae resistant to the toxin, providing further evidence that chitin is the cellular receptor for the P. inositovora toxin. Orf4p of pPin1-3 displays only weak similarities to the gamma-subunit of zymocin, which causes a G1 cell-cycle arrest in S. cerevisiae. However, disruption of the S. cerevisiae gene ELP3/TOT3, which encodes a histone-acetyltransferase that is essential for zymocin action, resulted in reduced sensitivity to the P. inositovora toxin also. Thus, despite obvious differences in genome organization and protein architecture, both killer systems very probably have similar modes of action.

Cis/trans isomerisation of unsaturated fatty acids in a cardiolipin synthase knock-out mutant of Pseudomonas putida P8.

The gene encoding cardiolipin synthase ( cls) from the phenol-degrading bacterium Pseudomonas putida P8, which rapidly adapts its membrane lipids to the presence of organic solvents by cis/trans isomerisation of unsaturated fatty acids, was isolated and completely sequenced. The functionality of the predicted gene product was proven by constructing a knock-out mutant that was significantly reduced in its growth rate both at elevated temperatures and in the presence of membrane-active solvents. Though the mutant showed a clear phenotype it was still able to synthesise trace amounts of cardiolipin. As an increase in cardiolipin (diphosphatidylglycerol) content is known to function as a long term membrane adaptation mechanism in pseudomonads, we tested whether the mutant compensates for the lack of the Cls by increased cis/trans isomerisation of unsaturated fatty acids. Increase in cis/trans isomerisation of unsaturated fatty acids was observed for the mutant at zero and low concentrations of 4-chlorophenol; however, cis/trans isomerisation is not able to fully compensate for the lack of cardiolipin production. Possibly, other long-term adaptation mechanisms are instrumental in compensating for the missing cardiolipin synthesis. As the cis/trans isomerase is activated similarly in the mutant and the wildtype, cis/trans isomerisation and cardiolipin production do not display mutual dependency.

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic ascomycete Calonectria morganii.

Conidia of the phytopathogenic fungus Calonectria morganii were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, governed by a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. Agrobacterium tumefaciens-mediated transformation yielded stable hygromycin B-resistant clones (average number (106 per 10(7)) [corrected] conidia). Putative transformants appeared to be mitotically and meiotically stable. The presence of the hph gene was checked by PCR. In four randomly chosen transformants, single-copy integrations of the marker gene at different chromosomal sites were proven by Southern analysis.

Saccharomyces cerevisiae cell wall chitin, the Kluyveromyces lactis zymocin receptor.

The exozymocin secreted by Kluyveromyces lactis causes sensitive yeast cells, including Saccharomyces cerevisiae, to arrest growth in the G(1) phase of the cell cycle. Despite its heterotrimeric (alpha beta gamma) structure, intracellular expression of its smallest subunit, the gamma-toxin, is alone responsible for the G(1) arrest. The alpha subunit, however, has a chitinase activity that is essential for holozymocin action from the cell exterior. Here we show that sensitive yeast cells can be rescued from zymocin treatment by exogenously applying crude chitin preparations, supporting the idea that chitin polymers can compete for binding to zymocin with chitin present on the surface of sensitive yeast cells. Consistent with this, holozymocin can be purified by way of affinity chromatography using an immobilized chitin matrix. PCR-mediated deletions of chitin synthesis (CHS) genes show that most, if not all, genetic scenarios that lead to complete loss (chs3 Delta), blocked export (chs7 Delta) or reduced activation (chs4 Delta), combined with mislocalization (chs4 Delta chs5 Delta; chs4 Delta chs6 Delta; chs4 Delta chs5 Delta chs6 Delta) of chitin synthase III activity (CSIII), render cells refractory to the inhibitory effects of exozymocin. In contrast, deletions in CHS1 and CHS2, which code for CSI and CSII, respectively, have no effect on zymocin sensitivity. Thus, CSIII-polymerized chitin, which amounts to almost 90% of the cell's chitin resources, appears to be the carbohydrate receptor required for the initial interaction of zymocin with sensitive cells.

Cloning, expression, and carbon catabolite repression of the bamM gene encoding beta-amylase of Bacillus megaterium DSM319.

The bamM gene from Bacillus megaterium DSM319 encoding an extracellular beta-amylase was isolated and completely sequenced. Chromosomal inactivation by deletion mutagenesis resulted in total loss of amylolytic activity, indicative of a single starch-degrading enzyme. Functional characterization of the expressed protein revealed a maltogenic enzyme exhibiting optimal activities at pH 7.5 and 50 degrees C. Amylase expression is subject to catabolite repression by glucose. A putative cis-acting catabolite-responsive element (CRE) was identified; it is located within the bamM coding region, matching the position of the predicted signal peptide processing site. Base substitutions introduced by site-directed mutagenesis within the bamM-CRE--retaining unchanged the amino acid sequence--provoked a remarkable relief from carbon catabolite repression (CCR), thereby proving functionality of the CRE for CCR.

Genome organization of the linear cytoplasmic element pPE1B from Pichia etchellsii.

The linear cytoplasmic element pPE1B from Pichia etchellsii CBS2011 (synonym Debaryomyces etchellsii) was totally sequenced. It consists of 12835 bp and has a remarkable high A+T content of 77.3%. The termini of pPE1B were found to consist of inversely orientated identical nucleotide repetitions 161 base pairs long, to which proteins are probably covalently linked at the 5' ends. Ten putative genes (open reading frames, ORFs) were identified, covering 96.5% of the total sequence. The predicted polypeptides correspond to proteins encoded by ORFs 2-11 of the linear plasmids pGKL2 of Kluyveromyces lactis and pSKL of Saccharomyces kluyveri. ORF1, existing on both latter elements, is lacking on pPE1B. An upstream conserved sequence motif (UCS) is located at the expected distance from the start codon of each of the 10 ORFs. As the arbitrarily chosen UCS6 was able to drive expression of a reporter gene in the heterologous pGKL-encoded killer system of K. lactis, extranuclear promoter function is probable. The almost congruent genome organization of pPE1B and other autonomous linear yeast plasmids sequenced so far, i.e. pGKL2 and pSKL, suggests a common, presumably viral, ancestor.

Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA-triphosphatase activities.

The predicted ORF3 polypeptide (Orf3p) of the linear genetic element pGKL2 from Kluyveromyces lactis was expressed in Bacillus megaterium as a fusion protein with a His(6X)-tag at the C-terminus for isolation by Ni-affinity chromatography. This is the first time that a yeast cytoplasmic gene product has been expressed heterologously as a functional protein in a bacterial system. The purified protein was found to display both RNA 5'-triphosphatase and guanylyltransferase activities. When the lysine residue present at position 177 of the protein within the sequence motif (KXDG), highly conserved in capping enzymes and other nucleotidyl transferases, was substituted by alanine, the guanylyltransferase activity was lost, thereby proving an important role for the transfer of GMP from GTP to the 5'-diphosphate end of the mRNA. Our in vitro data provides the first direct evidence that the polypeptide encoded by ORF3 of the cytoplasmic yeast plasmid pGKL2 functions as a plasmid-specific capping enzyme. Since genes equivalent to ORF3 of pGKL2 have been identified in all autonomous cytoplasmic yeast DNA elements investigated so far, our findings are of general significance for these widely distributed yeast extranuclear genetic elements.

Telomere sequences attached to nuclearly migrated yeast linear plasmid.

The yeast linear plasmid pCLU1, derived from pGKL1, has terminal proteins (TPs) covalently attached at the 5' ends of inverted terminal repeats (ITRs) and replicates in the cytoplasm, presumably using the TP as a primer for DNA synthesis. In Saccharomyces cerevisiae, under certain conditions, pCLU1 migrated into the nucleus and replicated in either linear or circular form. The linear-form plasmid lacked TPs; instead it carried host-telomere repeats at the ITR ends. The present study showed that (1) the added telomere was primarily composed of the repeated tracts of TGTGTGGGTGTGG, which was complementary to the RNA template of yeast telomerase, (2) the telomeric addition occurred at the very end of the ITRs, and (3) the sequence composition of the added telomeres was diverse among individual plasmids, but symmetrically identical at both ends of each plasmid. A similar mode of telomere addition was also observed in cells defective in the RAD52 gene.

Kluyveromyces lactis killer system: identification of a new gene encoded by pGKL2.

The cytoplasmic linear plasmid pGKL2 of the yeast Kluyveromyces lactis was reported to harbour ten open reading frames (ORF1-ORF10). By re-examining the sequence, we identified an additional ORF encoding a polypeptide of 70 amino acids; and a homologous ORF with 70% similarity was also identified on plasmid pSKL of Saccharomyces kluyveri. As for pGKL2, the newly detected ORF11 is located at the same position with the same direction. ORF11 transcripts of pGKL2 were verified by reverse transcriptase-polymerase chain reaction; and the corresponding promoter activity was proven by expression of a reporter gene driven by the ORF11 upstream conserved sequence, indicating that ORF11 constitutes a functional gene.

The URA5 gene encoding orotate-phosphoribosyl transferase of the yeast Kluyveromyces lactis: cloning, sequencing and use as a selectable marker.

A pair of degenerate primers was used for amplification and cloning of an internal fragment of the K. lactis URA5 gene. Primers were designed on the basis of highly conserved motifs within protein sequences predicted for URA5 genes from several microorganisms. Using the amplified fragment as a probe, we finally cloned and sequenced a 1.9 kb chromosomal fragment containing the orotate-phosphoribosyltransferase-encoding URA5 gene and an incomplete open reading frame strikingly similar to SEC65 of Saccharomyces cerevisiae and other yeasts, in which the gene encodes a subunit of the signal recognition particle. Uracil-requiring mutants of K. lactis CBS 683 were selected on media containing 5-fluoro-orotic acid and used as recipients in transformation experiments using K. lactis URA5 as the selectable marker, thereby proving functionality of the cloned gene.

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles.

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.

cis/trans isomerase of unsaturated fatty acids of Pseudomonas putida P8: evidence for a heme protein of the cytochrome c type.

From a pool of 600 temperature-sensitive transposon mutants of Pseudomonas putida P8, 1 strain was isolated that carries a mini-Tn5 insertion within the cytochrome c operon. As a result, genes involved in the attachment of heme to cytochrome c-type proteins are turned off. Accordingly, cytochrome c could not be detected spectrophotometrically. The mutant also exhibited a remarkable reduction of cis-trans isomerization capability for unsaturated fatty acids. Consistent with the genetic and physiological data is the detection of a cytochrome c-type heme-binding motif close to the N terminus of the predicted polypeptide of the cis/trans isomerase (cti) gene (CVACH; conserved amino acids in italics). The functional significance of this motif was proven by site-directed mutagenesis. A possible mechanism of heme-catalyzed cis-trans isomerization of unsaturated fatty acids is discussed.

Regulation of beta-galactosidase expression in Bacillus megaterium DSM319 by a XylS/AraC-type transcriptional activator.

The beta-galactosidase-encoding bgaM gene of Bacillus megaterium DSM319 and the divergently orientated bgaR operon were isolated and sequenced. Both traits are subject to catabolite repression. A set of single-gene replacement mutants was generated and used to analyze gene function. BgaR was found to be a XylS/AraC-type positive transcriptional regulator of bgaM; a potential regulator binding site overlaps the bgaM promoter. A mechanism for regulation of beta-galactosidase expression in B. megaterium is proposed.

Kluyveromyces lactis killer plasmid pGKL2: evidence for a viral-like capping enzyme encoded by ORF3.

ORF3 of the cytoplasmic linear plasmid pGKL2 was disrupted in vivo by integration of a selectable marker. Long-term cultivation of transformants carrying hybrid plasmids with a disrupted ORF3 under selective pressure did not deprive strains of the native counterpart, thereby proving its essentiality for pGKL2 replication and maintenance. The predicted ORF3 polypeptide was found to contain conserved motifs acquainted with mRNA-capping enzymes in the required order, just as in cytoplasmic viruses; new conserved motifs were also identified.

cis-trans isomerization of unsaturated fatty acids: cloning and sequencing of the cti gene from Pseudomonas putida P8.

Transposon mutants of Pseudomonas putida P8 were generated by applying a mini-Tn5 mutagenesis system. The mutants obtained were checked for their ability to tolerate increased temperatures and elevated phenol concentrations. Approximately 5,800 transposon mutants were used to generate a pool of 600 temperature-sensitive strains; one of these strains was identified as being damaged in its ability to perform cis-trans isomerization of fatty acids. A gene library of P. putida P8 was constructed and screened by using as a probe sequences immediately adjacent to the mini-Tn5 insertion. A DNA fragment that complemented the mutation was isolated and cloned. The corresponding gene, termed cti, is located close to the methionine synthase locus (metH) in P. putida P8. A cti-carrying fragment integrated into a plasmid also conferred the ability for cis-trans isomerization to Escherichia coli; the cti gene was completely sequenced, and the amino acid sequence was deduced.

Microbial linear plasmids.

While plasmids were originally considered to be generally circular until almost two decades ago, linear elements were reported to exist as well. They are now known to be common genetic elements in both, pro- and eukaryotes. Two types of linear plasmids exist, the so-called hairpin plasmids with covalently closed ends and those with proteins bound to their 5' termini. Hairpin plasmids are common in human-pathogenic Borrelia spirochetes, in which they are instrumental in escape from the immunological response; cryptic hairpin elements are present in mitochondria of the plant pathogenic fungus Rhizoctonia solani. Plasmids with 5' attached proteins constitute the largest group. In actinomycetous bacteria they are conjugative and usually confer advantageous phenotypes, e.g. formation of antibiotics, degradation of xenobiotics, heavy-metal resistance and growth on hydrogen as the sole energy source. In contrast, the majority of linear plasmids from eukaryotes are cryptic, with only a few exceptions. In some yeasts a killer phenotype may be associated, the most thoroughly investigated elements being those from Kluyveromyces lactis killer strains. In Neurospora spp. and in Podospora anserina, senescence and longevity respectively are correlated with linear plasmids. This review focuses on the biology of linear plasmids, their environmental significance and their use as tools in molecular and applied microbiology.