EMBL Accession Research Articles

Occurrence of natural dixenic associations between the symbiont Photorhabdus luminescens and bacteria related to Ochrobactrum spp. in tropical entomopathogenic Heterorhabditis spp. (Nematoda, Rhabditida).

Bacteria naturally associated with the symbiont Photorhabdus luminescens subsp. akhurstii were isolated from the entomopathogenic nematode Heterorhabditis indica. Bacterial isolates distinct from P. luminescens subsp. akhurstii were obtained from 33% of the samples. Fourteen bacterial isolates, from nematodes collected from three different Caribbean islands, were characterized by conventional phenotypic tests, restriction fragment length polymorphism and sequence analyses of PCR-amplified 16S rRNA genes (16S rDNAs). Isolates were grouped into three genotypes, each one being associated with one Caribbean island. Phenotypic characteristics and 16S rDNA analysis showed that the Photorhabdus-associated bacteria were closely related to Ochrobactrum anthropi for the group from Guadeloupe, and to Ochrobactrum intermedium for the two groups from the Dominican Republic and Puerto Rico. No pathogenicity of the Ochrobactrum spp. to the insects Galleria mellonella and Spodoptera littoralis (Lepidoptera) was detected. Since Ochrobactrum spp. are considered as human opportunist pathogens, the mass production of entomopathogenic nematodes for biological control requires strict vigilance.

A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon.

A 9.2 kb cryptic Mycobacterium fortuitum plasmid, pMF1, was isolated from strain 110 and its restriction map constructed. A 4.2 kb HindIII fragment of pMF1 was found to support replication in mycobacteria and this fragment was cloned and sequenced to characterize the replication elements of the plasmid. Computer analysis identified a putative Rep protein (362 amino acids) with high homology to the putative Rep protein of the Mycobacterium celatum plasmid pCLP and limited homology, mostly in the N-terminal region, to the Rep proteins of Mycobacterium avium pLR7, M. fortuitum pJAZ38 and Mycobacterium scrofulaceum pMSC262. A region containing a putative ori site was located upstream of the rep gene; this region displayed high homology at the nucleotide level with the predicted ori of pCLP and pJAZ38. A plasmid carrying the 4.2 kb HindIII fragment and a kanamycin resistance marker, designated pBP4, was maintained as a single-copy plasmid in Mycobacterium smegmatis and was stably inherited in the absence of antibiotic selection. Plasmid pBP4 was incompatible with the pJAZ38 replicon but was compatible with the widely used pAL5000 replicon, indicating that among the mycobacterial vectors now available there are two incompatibility groups. Significantly, the plasmid was able to replicate in the pathogen Mycobacterium tuberculosis, making it a useful tool for gene expression studies. To provide a choice of restriction sites and easy manipulation, a 2.1 kb fragment containing the minimal replication region was cloned to make the mycobacterial shuttle vector pBP10, which showed similar stability to pBP4.

Genes for the synthesis of the osmoprotectant glycine betaine from choline in the moderately halophilic bacterium Halomonas elongata DSM 3043, USA.

The genes involved in the oxidative pathway of choline to glycine betaine in the moderate halophile Halomonas elongata DSM 3043 were isolated by functional complementation of an Escherichia coli strain defective in glycine betaine synthesis. The cloned region was able to mediate the oxidation of choline to glycine betaine in E. coli, but not the transport of choline, indicating that the gene(s) involved in choline transport are not clustered with the glycine betaine synthesis genes. Nucleotide sequence analysis of a 4.6 kb segment from the cloned DNA revealed the occurrence of three ORFs (betIBA) apparently arranged in an operon. The deduced betI gene product exhibited features typical for DNA-binding regulatory proteins. The deduced BetB and BetA proteins showed significant similarity to soluble glycine betaine aldehyde dehydrogenases and membrane-bound choline dehydrogenases, respectively, from a variety of organisms. Evidence is presented that BetA is able to oxidize both choline and glycine betaine aldehyde and therefore can mediate both steps in the synthesis of glycine betaine.

Sequence analysis of two cosmids from Schizosaccharomyces pombe chromosome III.

We report the complete sequence of two cosmids, SPCC895 (38457 bp insert, EMBL Accession No. AL035247) and SPCC1322 (42068 bp insert, EMBL Accession No. AL035259), localized on chromosome III of the Schizosaccharomyces pombe genome. Fourteen Coding DNA sequences (CDSs) were identified in SPCC895 and 17 in SPCC1322. Two known genes were found in each cosmid: map2 and gms1 on SPCC895, encoding the mating type P-factor precursor and an UDP-galactose transporter, respectively, and bub1 and ade6 in SPCC1322, encoding a protein kinase and a phosphoribosylaminoimidazole carboxylase, respectively. The fission yeast K RNA gene has been localized to SPCC895. Three ribosomal proteins have been predicted among these two cosmids. Nine CDSs similar to known proteins were found on SPCC895, and seven on SPCC1322. They include putative genes for an uridylate kinase, a proteasome catalytic component, an ion transporter, a checkpoint protein, a translation initiation protein, a SNARE complex protein, a protein involved in cytoskeletal organization, a spindle pole body-associating protein, pre-mRNA splicing factor RNA helicase, a 3'-5' exonuclease for RNA 3' ss-tail, an UTP-glucose-1-phosphate uridylyltransferase, a leukotriene A(4) hydrolase, a member of the RanBP7-importin beta-Cse1p superfamily, a Ca(++)-calmodulin-dependent serine/threonine protein kinase and a prohibitin antiproliferative protein. One CDS is predicted to be an integral membrane protein. One CDS from SPCC895 is similar to a CDS of unknown function from Saccharomyces cerevisiae and three from SPCC1322 are similar to CDSs of unknown function from Candida albicans, S. cerevisiae and Sz. pombe, respectively. Finally, one CDS of SPCC895 and three of SPCC1322 correspond to orphan genes.

Functional cloning of the adenylate cyclase gene ofCandida albicans inSaccharomyces cerevisiae within a genomic fragment containing five other genes, including homologues ofCHS6 andSAP185

We have cloned a genomic fragment of Candida albicans by complementation of a Saccharomyces cerevisiae cyr1 mutant. This fragment contains the two-thirds C-terminal part of the adenylate cyclase CaCYR1. The complete gene has been sequenced from PCR fragments amplified from genomic DNA, and contains an ORF of 1690 amino acids closely related to other fungal adenylate cyclases. Adjacent to the adenylate cyclase gene, we have sequenced six other putative genes. CaCHS6, CaYNL191 and CaYJL098 are named on the basis of their close similarity with S. cerevisiae genes. ORFs CaYJL097a and CaYJL097b represent two repeated homologues of the S. cerevisiae YJL097w, which probably arose from an ancient duplication. The last one is a hypothetical ORF, CaYKR049, which presents only a very weak similarity with YKR049. The S. cerevisiae homologues of three of these genes are co-localized on chromosome X but with a different order and orientation.

Alastrim Smallpox Variola Minor Virus Genome DNA Sequences

Alastrim variola minor virus, which causes mild smallpox, was first recognized in Florida and South America in the late 19th century. Genome linear double-stranded DNA sequences (186,986 bp) of the alastrim virus Garcia-1966, a laboratory reference strain from an outbreak associated with 0.8% case fatalities in Brazil in 1966, were determined except for a 530-bp fragment of hairpin-loop sequences at each terminus. The DNA sequences (EMBL Accession No. Y16780) showed 206 potential open reading frames for proteins containing ≥60 amino acids. The amino acid sequences of the putative proteins were compared with those reported for vaccinia virus strain Copenhagen and the Asian variola major strains India-1967 and Bangladesh-1975. About one-third of the alastrim viral proteins were 100% identical to correlates in the variola major strains and the remainder were ≥95% identical. Compared with variola major virus DNA, alastrim virus DNA has additional segments of 898 and 627 bp, respectively, within the left and right terminal regions. The former segment aligns well with sequences in other orthopoxviruses, particularly cowpox and vaccinia viruses, and the latter is apparently alastrim-specific.

PseudoBase: a database with RNA pseudoknots.

PseudoBase is a database containing structural, functional and sequence data related to RNA pseudo-knots. It can be reached at http://wwwbio. Leiden Univ.nl/ approximately Batenburg/PKB.html. This page will direct the user to a retrieval page from where a particular pseudoknot can be chosen, or to a submission page which enables the user to add pseudoknot information to the database or to an informative page that elaborates on the various aspects of the database. For each pseudoknot, 12 items are stored, e.g. the nucleotides of the region that contains the pseudoknot, the stem positions of the pseudoknot, the EMBL accession number of the sequence that contains this pseudoknot and the support that can be given regarding the reliability of the pseudoknot. Access is via a small number of steps, using 16 different categories. The development process was done by applying the evolutionary methodology for software development rather than by applying the methodology of the classical waterfall model or the more modern spiral model.

Occurrence of two serotypes of sweet potato chlorotic stunt virus in East Africa and their associated differences in coat protein and HSP70 homologue gene sequences

Amongst samples of the sweet potato chlorotic stunt crinivirus (SPCSV) obtained from crops of sweet potato in Uganda, two serotypes (SEA1 and SEA2) were distinguished using a panel of monoclonal antibodies (Mabs) to a Kenyan isolate of SPCSV. SEA1 serologically resembled the Kenyan isolate of SPCSV whereas SEA2 has not previously been reported. SEA1 was predominant in eastern Uganda whereas SEA2 was predominant in southern and western Uganda. SEA2 tended to occur in more severely diseased sweet potato plants than SEA1. RNA was extracted from eight plants and replicate clones representing the heat shock protein 70 (HSP70) homologue and coat protein (CP) genes were generated by reverse transcription and PCR. Sequence analyses revealed substitutions at two nucleotide positions in the HSP70 homologue gene, although neither affected deduced amino‐acid sequences. Nucleotide substitutions in the CP gene region, which led to 11 amino‐acid substitutions, revealed two major groupings plus other minor variants.The EMBL accession numbers of the sequences reported in this paper are AJ010754 through to AJ01769 (coat protein sequences) and AJ010914 through to AJ010929 (partial HSP70 homologue gene sequences).

Cloning and characterization of the EXG1 gene from the yeast Yarrowia lipolytica.

The YlEXG1 gene of Yarrowia lipolytica, encoding an exo-1, 3-beta-glucanase, was isolated by screening a genomic library with a DNA probe obtained by PCR amplification, using oligonucleotides designed according to conserved regions in the EXG1, EXG2 and SSG1 genes from Saccharomyces cerevisiae. YlEXG1 consists of a 1263 bp open reading frame encoding a protein of 421 amino acids with a calculated molecular weight of 48 209 Da. Northern blot analysis revealed a unique YlEXG1-specific transcript, 1.4 kb long. A putative pre(signal)-peptide of 15 amino acids is proposed at the N-terminal domain of the primary translation product. The deduced amino acid sequence shares a high degree of homology with exo-1, 3-beta-glucanases from other yeast species, including S. cerevisiae, Kluyveromyces lactis, Pichia angusta and Debaryomyces occidentalis. YlExg1p contains the invariant amino acid positions which have been shown to be important in the catalytic function of family 5 glycosyl hydrolases. Chromoblot analysis indicated that YlEXG1 is located on chromosome VI. Disruption of YlEXG1 did not result in a phenotype under laboratory conditions and did not prevent the yeast-hypha transition. The sequence data reported in this paper have been assigned EMBL Accession No. Z46872.

Genetic and functional analysis of genes required for the post-modification of the polyketide antibiotic TA of Myxococcus xanthus.

The antibiotic TA of Myxococcus xanthus is a complex macrocyclic polyketide, produced through successive condensations of acetate by a type I PKS (polyketide synthase) mechanism. The genes encoding TA biosynthesis are clustered on a 36 kb DNA fragment, which has been cloned and analysed. The chemical structure of TA and the mechanism by which it is synthesized indicate the need for several post-modification steps, which are introduced into the carbon chain of the polyketide to form the final bioactive molecule. These include the addition of several carbon atoms originating from acetate carbonyl, three C-methylations, O-methylation and a specific hydroxylation. This paper reports the analysis of five genes which are involved in the post-modification of TA. Their functional analysis, by specific gene disruption, suggests that they may be essential for the production of the active antibiotic. The characteristics and organization of the genes suggest that they may be involved in the addition of the carbon atoms which arise from acetate.

Sequence analysis of three Bacillus cereus loci carrying PIcR-regulated genes encoding degradative enzymes and enterotoxin.

PIcR is a pleiotropic regulator of extracellular virulence factors in the opportunistic human pathogen Bacillus cereus and the entomopathogenic Bacillus thuringiensis, and is induced in cells entering stationary phase. Among the genes regulated by PIcR are: pIcA, encoding phosphatidylinositol-specific phospholipase C (PI-PLC); plc, encoding phosphatidylcholine-preferring phospholipase C (PC-PLC); nhe, encoding the non-haemolytic enterotoxin; hbl, encoding haemolytic enterotoxin BL (HBL); and genes specifying a putative S-layer like surface protein and a putative extracellular RNase. By analysing 37.1 kb of DNA sequence surrounding hbl, plcA and plcR, 28 ORFs were predicted. Three novel genes putatively regulated by PlcR and encoding a neutral protease (NprB), a subtilase family serine protease (Sfp) and a putative cell-wall hydrolase (Cwh) were identified. The corresponding sfp and cwh genes were located in the immediate upstream region of plcA and could both be regulated by a putative PlcR-binding site positioned between the inversely transcribed genes. Similarly, nprB was positioned directly upstream and transcribed in the opposite orientation to plcR. Genes surrounding plcA, plcR and hblCDAB that were lacking an upstream PlcR regulatory sequence did not appear to serve functions apparently related to PlcR and did not exhibit a conserved organization in Bacillus subtilis.

Kluyveromyces lactis HIS4 transcriptional regulation: similarities and differences to Saccharomyces cerevisiae HIS4 gene

Sequence analysis of the Kluyveromyces lactis HIS4 ( KlHIS4) gene promoter reveals relevant differences in comparison to the Saccharomyces cerevisiae HIS4 homologous gene. Among them are the absence of a Rap1 binding site and the presence of only three putative Gcn4 binding consensus sites instead of the five described in the S. cerevisiae promoter. Since these factors are implicated in the general control, we investigated the transcriptional regulation of the KlHIS4 gene under conditions of amino acid starvation and discovered that the mechanisms previously described for S. cerevisiae HIS4 regulation and related to general control are not functional in K. lactis. The expression analysis of the KlHIS4 gene under phosphate starvation or high adenine supply shows that factors, such as Bas1 or Bas2, involved in the basal control may also operate in a different way in K. lactis. Interestingly, and also in contrast to the HIS4 regulation in S. cerevisiae, we found domains for Nit2-like and yeast-Ap1-like binding sequences. Northern analyses showed transcriptional activation under ammonia starvation and oxidative stress. The EMBL accession number for the KlHIS4 promoter is AJ238494.

Nitrogen metabolism in Streptomyces coelicolor A3(2): modification of glutamine synthetase I by an adenylyltransferase.

An internal adenylyltransferase gene (glnE) fragment from Streptomyces coelicolor was amplified using heterologous PCR primers derived from consensus motifs. The sequence had significant similarity to bacterial glnE genes, and included a motif typical of the C-terminal adenylyltransferase domain of glnE. glnE from S. coelicolor lies on the Asel-C fragment of the chromosome and is localized near glnA (encoding glutamine synthetase I, GSI) and glnII (encoding GSII). To analyse the function of glnE in S. coelicolor, glnE (S. coelicolor E4) and glnA (S. coelicolor HT107) gene replacement mutants were constructed. The GSI activity of the glnE mutant was not down-regulated after an ammonium shock. However, the GSI activity of the wild-type cells decreased to 60% of the original activity. The glnA mutant is not glutamine auxotrophic, but in the gamma-glutamyltransferase assay no GSI activity was detected in unshifted and shifted HT107 cells. By snake venom phosphodiesterase treatment the GSI activity in the wild-type can be reconstituted, whereas no alteration is observed in the E4 mutant. Additionally, the loss of short-term GSI regulation in the E4 mutant was accompanied by an increased glutamine:glutamate ratio.

Disruption of sblA in Streptomyces lividans permits expression of a heterologous alpha-amylase gene in the presence of glucose.

In a transposition mutant of Streptomyces lividans TK24, the usually glucose-repressible expression of a heterologous alpha-amylase gene (aml) became resistant to glucose repression. The transposon had inserted into an ORF called sblA which encodes a 274 aa product sharing significant sequence similarities with various phosphatases that act on small phosphorylated substrates. sblA was transcribed as a monocistronic mRNA and its transcription was enhanced at the transition phase. Because its transcriptional and putative translational start points coincide, sblA is likely to be translated in the absence of a conventional RBS. The sblA-disrupted mutant is characterized by early growth arrest in glucose-grown cultures and by partial relief of glucose repression of aml expression.

Isolation of Trichoderma harzianum Th2 from Commercial Mushroom Compost in Spain.

Trichoderma spp. are associated with green mold of mushrooms. This fungal disease has caused severe losses in mushroom production in countries such as Ireland, the United Kingdom, Canada, and the United States. This disease is caused by two biotypes of T. harzianum (Th2, Europe; Th4, North America) (1,2). Both biotypes have not been detected in mushrooms or other material in Spain previously. However, during 1998, green mold was detected at facilities dedicated to produce compost, as well as in facilities used to produce Agaricus bisporus (Lange) Imbach. Three compost samples were isolated from commercial bags with mushroom substrate and three more samples were taken from mushroom yards. Several spores were isolated by the dilution plate method. Initial identification of the pathogenic fungi was made by examining cultures grown on potato dextrose agar. Morphological characteristics of all isolates coincided with the description of T. harzianum (3). The following amounts of CFU per g were found in commercial compost samples: 1.2 × 108, 5.5 × 107, and 1.4 × 107 per g; whereas 3 × 108, 12.4 × 107, and 2.2 × 106 were obtained from mushroom yards. The fragment containing the internal transcribed spacer (ITS1) was amplified and sequenced for each of the six samples obtained. The ITS1 sequence (201 bp) was identical in all samples, and the sequence was aligned, with Clustal W, with Th2 and Th4 biotype sequences of the EMBL data base. The ITS1 sequence showed 0.55% divergence from Th2 isolates and more distance, 3.3%, with Th4 isolates. The ITS1 sequence obtained with all Spanish samples studied, EMBL accession number AJ1321550, was identical to that described for the Irish isolate Th2I (#63), with accession number U78880 in the EMBL data base (1). This is the first description of the Th2 biotype in Spain.

The KlPMR1 gene of Kluyveromyces lactis encodes for a P-type Ca(2+)-ATPase.

A novel P-type Ca(2+)-ATPase gene has been cloned and sequenced in the yeast Kluyveromyces lactis. The gene has been named KlPMR1 and is localized on chromosome I. The putative gene product contains 936 residues and has a calculated molecular weight of 102,437 Da. Analysis of deduced amino acid sequence (KlPmr1p) indicated that the encoded protein retains all the highly conserved domains characterizing the P-type ATPases. KlPmr1p shares 71% amino acid identity with Pmr1p of S. cerevisiae, 62% with HpPmr1p of Hansenula polymorpha, 56% with Y1Pmr1p of Yarrowia lipolytica and 52% with the Ca(2+)-ATPase encoded for by the SPCA1 gene of Rattus norvegicus; these similarities place KlPmr1p in the SPCA group (secretory pathway Ca(2+)-ATPase) of the P-type ATPases. The K. lactis strain harbouring the Klpmr1 disrupted gene is not able to grow in presence of low calcium concentrations and shows hypersensitivity to high concentrations of EGTA in the medium. These defects are relieved by PMR1 of S. cerevisiae on a centromeric plasmid, demonstrating that KlPMR1 encodes for a functional Pmr1p homologue.

The porcine poly(rC)-binding protein 2 (PCBP2) gene maps to chromosome 5.

Description: A recombinant clone was isolated at random from a skeletal muscle cDNA library1. The sequence of the resulting 598 bp insert (EMBL accession number X94253) was compared in EMBL and GenBank databases using BLASTN2. The comparisons showed the highest matches with mouse and human PCBP2 cDNAs3−5 (mouse X75947, mouse X97982, human X78136and mouse L19661; BLASTN probability were 1·9e-182, 1·1e-176, 1·6e-174 and 4·7 e-172, respectively). Excluding two gaps that could be due to alternative splicing, the deduced porcine protein sequence revealed the complete identity with corresponding human ( X78136) and mouse ( L19661) sequences. The mouse sequences X75947and X97982showed only one amino acid difference (Arg instead of Gly) with the deduced amino acid sequence of the porcine cDNA clone isolated.

Regulation of the expression of the Kluyveromyces lactis PDC1 gene: carbon source-responsive elements and autoregulation.

The yeast Kluyveromyces lactis has a single structural gene coding for pyruvate decarboxylase (KIPDC1). In order to study the regulation of the expression of KIPDC1, we have sequenced (EMBL Accession No. Y15435) its promoter and have fused the promoter to the reporter gene lacZ from E. coli. Transcription analysis in a Klpdc1 delta strain showed that KIPDC1 expression is subject to autoregulation. The PDC1 gene from Saccharomyces cerevisiae was able to complement the Rag- phenotype of the Klpdc1 delta mutant strain and it could also repress transcription of the KIPDC1-lacZ fusion on glucose. A deletion analysis of the promoter region was performed to study carbon source-dependent regulation and revealed that at least two cis-acting regions are necessary for full induction of gene expression on glucose. Other cis-elements mediate repression on ethanol.

Translation elongation factor 2 is encoded by a single essential gene in Candida albicans.

Translation elongation factor 2 (eEF2) is a large protein of more than 800 amino acids which establishes complex interactions with the ribosome in order to catalyze the conformational changes needed for translation elongation. Unlike other yeasts, the pathogenic fungus Candida albicans was found to have a single gene encoding this factor per haploid genome, located on chromosome 2. Expression of this locus is essential for vegetative growth, as evidenced by placing it under the control of a repressible promoter. This C. albicans gene, named EFT2, was cloned and sequenced (EMBL accession number Y09664). Genomic and cDNA sequence analysis identified common transcription initiation and termination signals and an 842 amino acid open reading frame (ORF), which is interrupted by a single intron. Despite some genetic differences, CaEFT2 was capable of complementing a Saccharomyces cerevisiae Deltaeft1 Deltaeft2 null mutant, which lacks endogenous eEF2, indicating that CaEFT2 can be expressed from its own promoter and its intron can be correctly spliced in S. cerevisiae.

Cloning and characterization of the human novel gene encoding 6.2 kDa protein highly expressed upon ultraviolet irradiation in DNA-PK-deleted human glioma M059J cells.

The full length of a gene isolated using a methylated DNA binding column as previously reported (Cross et al., 1994) has been cloned. The gene encodes 55 amino acids (6.2 kDa), and has a transcript of approximately 0.45 kb. It has 2 exons in the genome. After searching for sequence homology (BLAST), a homology to hypothetically 6.3 kDa protein located at cosmid ZK652 (protein P34660; EMBL Accession No. L14429) of C. elegans chromosome 3 was noted (51.9% amino acid homology). It has 1 site (-SQ-) for DNA-dependent protein kinase (DNA-PK) phosphorylation and 2 sites of myristilation. This novel gene is very strongly expressed (about 25.6 times) in DNA-PK-deleted human glioma cell line (M059J) 2 hrs after ultraviolet (UV) irradiation compared to the DNA-PK +/+ cell line (M059K) which expresses the gene at a relatively low level (about 7.5 times). Therefore, this result suggests that DNA-PK may negatively regulate the transcriptional level of this gene in M059K cells upon UV damage (50 J/m2), associated with cell toxicity.