Amino Acid Sequence Of ORF Research Articles

The ORF1 Protein Encoded by LINE‐1: Structure and Function During L1 Retrotransposition

LINE-1, or L1 is an autonomous non-LTR retrotransposon in mammals. Retrotransposition requires the function of the two, L1-encoded polypeptides, ORF1p and ORF2p. Early recognition of regions of homology between the predicted amino acid sequence of ORF2 and known endonuclease and reverse transcriptase enzymes led to testable hypotheses regarding the function of ORF2p in retrotransposition. As predicted, ORF2p has been demonstrated to have both endonuclease and reverse transcriptase activities. In contrast, no homologs of known function have contributed to our understanding of the function of ORF1p during retrotransposition. Nevertheless, significant advances have been made such that we now know that ORF1p is a high affinity RNA binding protein that forms a ribonucleoprotein particle together with L1 RNA. Furthermore, ORF1p is a nucleic acid chaperone and this nucleic acid chaperone activity is required for L1 retrotransposition.

Characterization of a small cryptic plasmid, pHP51, from a Korean isolate of strain 51 of Helicobacter pylori

The nucleotide sequence of a 3955-bp Helicobacter pylori plasmid, pHP51 was determined, and two open reading frames, ORF1 and ORF2, were identified. The deduced amino acid sequence of ORF1 was highly conserved (87–89%) among plasmid replication initiation proteins, RepBs. The function of ORF2 was not assigned because it lacked known functional domains or sequence similarity with other known proteins, although it had a HPFXXGNG motif that was also found in the cAMP-induced filamentation ( fic) gene. Three kinds of repeats were present on the plasmid outside of the ORFs, including the R1 and R2 repeats that are common in H. pylori plasmids. One 100-bp sequence detected in the noncoding region of pHP51 was highly similar to the genomic sequence of H. pylori 26695.

Characterization of aldehyde ferredoxin oxidoreductase gene defective mutant in Magnetospirillum magneticum AMB-1

A non-magnetic mutant of Magnetospirillum magneticum AMB-1, designated as NMA21, was generated by mini-Tn5 transposon mutagenesis to identify genes involved in bacterial magnetic particle (BMP) synthesis. Alignment of the DNA sequences flanking the transposon allowed the isolation of an open reading frame (ORF2) within an operon consisting of five genes. The amino acid sequence of ORF2 showed homology with tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus (48% identity and 64% similarity), which functions for aldehyde oxidation. AOR was found to be expressed under microaerobic conditions and localized in the cytoplasm of AMB-1. Iron uptake and growth of NMA21 were lower than wild type. Transmission electron microscopy (TEM) of NMA21 revealed that no BMPs were completely synthesized, but polyhydroxybutyrate (PHB)-like granules were persistently produced. These results indicate that AOR may contribute to ferric iron reduction during BMP synthesis in M. magneticum AMB-1 under microaerobic respiration.

Isolation and characterization of the glnD gene of Gluconacetobacter diazotrophicus, encoding a putative uridylyltransferase/uridylyl-removing enzyme

The glnD gene of Gluconacetobacter diazotrophicus was isolated by complementation of the Azotobacter vinelandii glnD ( nfrX) mutant strain MV17 using a pLAFR3 cosmid library. The 5 kb chromosomal DNA region encoding the glnD gene on cosmid pAD401 was identified by introduction of deletions as well as subcloning of restriction fragments followed by subsequent DNA sequencing. Three open reading frames were identified with the deduced amino acid sequence of ORF1 showing significant homologies to known GlnD proteins of other proteobacteria such as Sinorhizobium meliloti, Rhizobium tropici, Escherichia coli and Azotobacter vinelandii. A mutagenesis of the chromosomal glnD gene was carried out by insertion of an interposon carrying the kanamycin resistance gene of Tn 5. Mutants carrying the cassette inserted into a central region of glnD could not be isolated, while an interposon mutation at the 3′ end of glnD was successful. The resulting strain showed a prolonged generation time in complex growth medium and was unable to utilize ammonium as sole nitrogen source. This phenotype appears to be pleiotropic, since the addition of single amino acids to the minimal medium was not sufficient to allow growth. Furthermore, the glnD mutant was able to express nitrogenase under diazotrophic as well as repressing growth conditions.

Biosynthesis of sulfur-containing amino acids in Streptomyces venezuelae ISP5230: roles for cystathionine beta-synthase and transsulfuration.

A 0.5 kb fragment of Streptomyces venezuelae ISP5230 genomic DNA was amplified by PCR using primers based on consensus sequences of cysteine synthase isozyme A from bacteria. The deduced amino acid sequence of the PCR product resembled not only cysteine synthase sequences from prokaryotes and eukaryotes but also eukaryotic cystathionine beta-synthase sequences. Probing an Str. venezuelae genomic library with the PCR product located a hybridizing colony from which pJV207 was isolated. Sequencing and analysis of the Str. venezuelae DNA insert in pJV207 detected two ORFs. The deduced amino acid sequence of ORF1 matched both cysteine synthase and cystathionine beta-synthase sequences in GenBank, but its size favoured assignment as a cystathionine beta-synthase. ORF2 in the pJV207 insert was unrelated in function to ORF1; in its sequence the deduced product resembled acetyl-CoA transferases, but disruption of the ORF did not cause a detectable phenotypic change. Disruption of ORF1 failed to elicit cysteine auxotrophy in wild-type Str. venezuelae, but in the cys-28 auxotroph VS263 it prevented restoration of prototrophy with homocysteine or methionine supplements. The change in phenotype implicated loss of the transsulfuration activity that in the wild-type converts these supplements to cysteine. This study concludes that disruption of ORF1 inactivates a cbs gene, the product of which participates in cysteine synthesis by transsulfuration. Enzyme assays of Str. venezuelae mycelial extracts confirmed the formation of cysteine by thiolation of O-acetylserine, providing the first unambiguous detection of this activity in a streptomycete. Enzyme assays also detected cystathionine gamma-synthase, cystathionine beta-lyase and cystathionine gamma-lyase activity in the extracts and showed that the substrate for cystathionine gamma-synthase was O-succinyl-homoserine. Based on assay results, the cys-28 mutation in Str. venezuelae VS263 does not inactivate the cysteine synthase gene but impairs expression in cultures grown in minimal medium.

Shrimp Taura syndrome virus: genomic characterization and similarity with members of the genus Cricket paralysis-like viruses.

The single-stranded genomic RNA of Taura syndrome virus (TSV) is 10205 nucleotides in length, excluding the 3' poly(A) tail, and contains two large open reading frames (ORFs) that are separated by an intergenic region of 207 nucleotides. The ORFs are flanked by a 377 nucleotide 5' untranslated region (UTR) and a 226 nucleotide 3' UTR followed by a poly(A) tail. The predicted amino acid sequence of ORF1 revealed sequence motifs characteristic of a helicase, a protease and an RNA-dependent RNA polymerase, similar to the non-structural proteins of several plant and animal RNA viruses. In addition, a short amino acid sequence located in the N-terminal region of ORF1 presented a significant similarity with a baculovirus IAP repeat (BIR) domain of inhibitor of apoptosis proteins from double-stranded DNA viruses and from animals. The presence of this BIR-like sequence is the first reported in a single-stranded RNA virus, but its function is unknown. The N-terminal amino acid sequence of three TSV capsid proteins (55, 40 and 24 kDa) were mapped in ORF2, which is not in the same reading frame as ORF1 and possesses an AUG codon upstream of the structural genes. However, the intergenic region shows nucleotide sequence similarity with those of the genus Cricket paralysis-like viruses, suggesting a similar non-AUG-mediated translation mechanism. The structure of the TSV genome [5' UTR-non-structural proteins-intergenic UTR-structural proteins-3' UTR-poly(A) tail] is similar to those of small insect-infecting RNA viruses, which were recently regrouped into a new virus genus, Cricket paralysis-like viruses.

Similarity between Copper Resistance Genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato

Twenty-eight strains of Pseudomonas syringae pv. actinidiae isolated in 1984, 1987 and 1988 from kiwifruit orchards in Japan were tested for their resistance to copper sulfate. All strains isolated in 1984 were copper sensitive with a minimum inhibitory concentration (MIC) of cupric sulfate of 0.75 mM. However, some strains isolated in 1987 and 1988 were resistant, with the MIC ranging from 2.25 to 3.0 mM. All copper-resistant strains contained at least one of two plasmids, pPaCul (about 70.5 kb) or pPaCu2 (about 280 kb), or both. In a copper-resistant strain Pa429, the location of the copper-resistance gene(s) was examined by insertional inactivation with Tn5. The MIC of copper sulfate in the copper-sensitive mutant obtained by Tn5 tagging decreased from 2.75 to 0.75 mM. The 14.5 kb BamHI fragment, designated pPaCuB14, containing the same locus mutagenized with Tn5 was cloned from pPaCu1. However, pPaCuB14 did not confer copper resistance in the transformant of copper-sensitive strain Pa21R, suggesting that this clone did not contain a full set of copper-resistance gene(s). Then a cosmid library of pPaCu1 was constructed and six cosmid clones hybridized with pPaCuB14 were selected. One of the six cosmids, designated pPaCuC1, conferred a near wild-type level of copper resistance in the transformant of the copper-sensitive strain. pPaCuC1 had a homologous region that hybridized with all of the PCR-amplifled fragments of copA, copB, copR, and copS genes of P. syringae pv. tomato. DNA sequence analysis of the homologous region revealed the existence of four open reading frames (ORF A, B, R and S) oriented in the same direction. The predicted amino acid sequences of ORF A, B, R and S had 80, 70, 97 and 95% identity with CopA, B, R and S of P. syringae pv. tomato, respectively.

Molecular characterization of the Treponema denticola fliQ region.

A Treponema denticola 4.2 kb DNA region containing four complete genes (orfl, fliQ, fliR, and flhB) and a truncated gene (flhA') was sequenced and analyzed. The deduced amino acid sequences of FliQ, FliR, FlhB and FlhA' have significant homology with bacterial proteins associated with the flagellar export apparatus, whereas the deduced amino acid sequence of Orf1 has homology with an E. coli alcohol dehydrogenase. A putative sigma70-like promoter was identified upstream of fliQ. RT-PCR analysis indicated that fliQ, fliR, flhB and flhA' are co-transcribed independently of orfl, suggesting that the motility-associated genes are components of an operon. The location of the T. denticola fliQ-flhA' genes differs from that of the corresponding T. pallidum and Borrelia burgdorferi genes which are present in the large fla or flgB flagellar operons, respectively.

Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria.

The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6, 868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 x 10(6) CFU/microg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10(4) to 10(7) CFU/microg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.

Nucleotide sequence analysis of Triatoma virus shows that it is a member of a novel group of insect RNA viruses.

Triatoma virus (TrV) is the only virus described to date that infects triatomines, and has previously been considered to be a member of the family Picornaviridae on the basis of physico-chemical properties. The genome of TrV was sequenced completely (9010 nt). Analysis of the sequence revealed the presence of two large open reading frames (ORFs). The predicted amino acid sequence of ORF1 (nt 549-5936) showed significant similarity to the non-structural proteins of several animal and plant RNA viruses. This ORF product contains sequence motifs characteristic of RNA-dependent RNA polymerases (RdRp), cysteine proteases and RNA helicases. ORF1 is preceded by 548 nucleotides of non-coding RNA and the two ORFs are separated by 172 nucleotides of non-coding RNA. Direct N terminus sequence analysis of two capsid proteins showed that ORF2 (nt 6109-8715) encodes the structural proteins of TrV. The predicted amino acid sequence of ORF2 is very similar to the corresponding regions of Drosophila C virus, Plautia stali intestine virus, Rhopalosiphum padi virus and Himetobi P virus and to a partial sequence from the 3' end of the cricket paralysis virus genome. All of these viruses have a novel genome organization and it has been proposed that they are not members of the Picornaviridae, as previously thought, but belong to a new virus family. On the basis of similarities of genome organization, we propose that TrV also belongs to this new virus family.

Cloning, sequencing, and expression of three Bartonella henselae genes homologous to the Agrobacterium tumefaciens VirB region.

A 17-kDa, immunodominant antigen of Bartonella henselae Houston-1 has previously been cloned, sequenced, and characterized. This clone (H13) contains the 17-kDa antigen gene plus a partial open reading frame, designated ORF1, which is 459 nucleotides long and is directly upstream of the 17-kDa gene. Comparison of the deduced partial amino acid sequence of ORF1 with that of other known genes in GenBank revealed significant identity with several other bacterial virulence genes, including VirB4 of the Agrobacterium tumefaciens virB operon (56/149 amino acids). An overlapping clone, pGB3, was recovered and shown to contain a 3.0-kb region upstream of the 17-kDa gene. Sequence analysis revealed three ORFs upstream of the gene. The deduced amino acid sequence of each ORF was compared with sequences in GenBank, and identity was found with VirB2, VirB3, and VirB4 of A. tumefaciens. In vitro transcription/translation and SDS-PAGE demonstrated that three proteins of 9 kDa, 10 kDa, and 92 kDa, corresponding to the predicted molecular weight of 10.9 kDa, 11.7 kDa, and 89.9 kDa of VirB2, VirB3, and VirB4, respectively, could be expressed from these coding regions. These results indicate that virulence-associated genes and their overall chromosomal arrangement are relatively well conserved between B. henselae and other gram-negative bacteria such as A. tumefaciens.

Cloning, Sequencing, and Characterization of Two Clustered Cellulase-Encoding Genes, celS1 and celS2, from Streptomyces viridosporus T7A and their Expression in Escherichia coli.

A recombinant clone, pBLP, containing a 4.1kb fragment of Streptomyces viridosporus T7A chromosomal DNA was shown to confer endoglucanase activity in Escherichia coli cells. Further subcloning and sequence analysis revealed two co-transcribed Open Reading Frames (ORF) with high sequence similarities to other Streptomyces endoglucanase-encoding genes. A signal peptide, a catalytic domain and cellulose binding domain were identified within ORF1 (celS1). The amino acid sequence of ORF2 (celS2) showed similarity with cellulose binding protein (p40) of Streptomyces halstedii. The hydrophobic analysis of CelSl revealed that it belongs to the family H cellu-lase catalytic domain. The rare TTA codon encoding leucine was found in the signal sequence of both the cellu-lase ORFs, indicating translational dependence on the bldA gene product, a cognate tRNA responsible for translating the TTA codons. The presence of 14 bp inverted repeats in the 5’-end of these genes, was consistent with the highly conserved positive regulatory structure found in other endoglucanase genes.

Sequence analysis of the cryptic plasmid pMG101 from Rhodopseudomonas palustris and construction of stable cloning vectors.

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of alpha-proteobacteria.

A novel protein of Erysipelothrix rhusiopathiae that confers haemolytic activity on Escherichia coli.

Erysipelothrix rhusiopathiae, the cause of swine erysipelas and human erysipeloid, produces a haemolysin. A recombinant plasmid, pHLY, conferring haemolytic activity on Escherichia coli was isolated from a genomic library of Ery. rhusiopathiae strain Tama-96. This plasmid was stable in RecA- E. coli, but unstable in a RecA+ strain. A spontaneous deletion plasmid, pMini-HLY, also conferring haemolytic activity was derived from pHLY. Two ORFs were detected in pHLY. Analysis of pMini-HLY and other deletion clones established that ORF2 was associated with haemolytic activity. The sequence of ORF1 was highly homologous to those of transposases in the IS30 family. The deletion which generated pMini-HLY was between two short direct repeat (DR) sequences flanking the ORF1 sequence, and there were inverted repeat sequences inside the two DR sequences, suggesting an insertion element. No sequence homology to the deduced amino acid sequence of ORF2 was detected in the databases, but its sequence was characteristic of a surface lipoprotein. Western blot analysis, using antiserum against the 16 kDa protein produced from ORF2, found the protein to be commonly distributed in all Erysipelothrix species.

Nucleotide Sequence and Transcription Map of Porcine Adenovirus Type 3

The complete nucleotide sequence of porcine adenovirus type 3 was determined and a transcriptional map for the genome was constructed. The size of the genome is 34094 bp in length with an unusually high G + C content (63.7%), the highest thus far reported for any adenovirus. Overall organization of the genome is similar to that for previously sequenced adenoviral DNAs, but there also were distinct differences. The late regions genes are organized into six families, instead of five as they are in human adenovirus type 2. In contrast to bovine adenovirus type 3 and ovine adenovirus, which lack virion-associated RNA genes, the nucleotide sequence analysis of the viral genome indicates that it encodes one short VA RNA species. With the exception of the fiber and a 33-kDa nonstructural protein, the predicted amino acid sequences of the open reading frames in the late regions and the E2 region and IVa2 exhibited a high level of homology, whereas the deduced amino acid sequences of ORFs in E1, E3, and E4 regions, and the pIX showed a lesser homology with the corresponding proteins of other adenoviruses. The proteins V, VII, and IX are unusually long, and the protein VII lacks the consensus protease cleavage site. Genomic and cDNA sequence analysis has identified promoters, cap sites, intron–exon boundaries, polyadenylation signals, and polyadenylation sites in the viral genome.

Psittacine Beak and Feather Disease Virus Nucleotide Sequence Analysis and Its Relationship to Porcine Circovirus, Plant Circoviruses, and Chicken Anaemia Virus

Cloning and sequencing of the circular, single-stranded DNA of one isolate of psittacine beak and feather disease virus (BFDV) demonstrate a genome composed of a circular molecule of 1993 nucleotide bases. An analysis of the assembled replicative form demonstrated seven open reading frames (ORFs) (three in the virion strand and four in the complementary strand), potentially encoding seven viral proteins of >8.7 kDa. High amino acid sequence similarity was demonstrated between a potential 33.3-kDa protein product of ORF1 of BFDV and the replicase-associated protein of porcine circovirus (PCV), subterranean clover stunt virus, and faba bean necrotic yellows virus. However, significant similarity in nucleotide or amino acid sequences was not present between BFDV and chicken anaemia virus. A potential stem–loop structure similar to that found in PCV and plant circoviruses was present in the putative encapsidated strand of the BFDV genome. At the top of this structure, a nonanucleotide motif (TAGTATTAC) similar to that of PCV, plant circoviruses, and geminiviruses also was recognised. Comparison of the deduced amino acid sequences of ORF2 of BFDV and PCV demonstrated 29.1% identity, and in both viruses, ORF2 is located on the complementary strand, beginning close to or within the hairpin stem. Our findings provide further evidence of a close relationship among BFDV, PCV, and plant circoviruses but not chicken anaemia virus.

Tdd-3, a tRNA gene-associated poly(A) retrotransposon from Dictyostelium discoideum.

The full-length 5218-bp sequence of the mobile genetic element Tdd-3 from Dictyostelium discoideum is described. Tdd-3 encodes two overlapping open reading frames (ORFs) flanked by non-redundant, untranslated regions. The deduced amino acid sequence of ORF2 is homologous to reverse transcriptases (RTs) encoded by the class of poly(A) retrotransposons. ORF2 also encodes a putative protein domain related to the family of apurinic/apyrimidinic (AP) endonucleases, whose retroelement-encoded homologs have recently been proposed to represent the integrase function of poly(A) retrotransposons. Comparison of several genomic Tdd-3 copies revealed that element insertion is orientation specific and occurs about 100 bp downstream of tRNA genes in the D. discoideum genome. These properties of Tdd-3 suggest that the element is a tRNA gene-associated poly(A) retroelement present in the D. discoideum genome. Analysis of several cloned cDNAs derived from Tdd-3-specific plus strand RNAs indicate that the element is transcribed and polyadenylated during the growth of D. discoideum cells.

Nucleotide Sequence Analysis Shows thatRhopalosiphum padiVirus Is a Member of a Novel Group of Insect-Infecting RNA Viruses

Rhopalosiphum padivirus (RhPV) is an aphid virus that has been considered a member of thePicornaviridaebased on physicochemical properties. The 10,011-nt polyadenylated RNA genome of RhPV was completely sequenced. Analysis of the sequence revealed the presence of two open reading frames (ORFs). The predicted amino acid sequence of ORF1, representing the first 6600 nt of the RhPV genome, showed significant similarity to the nonstructural proteins of several plant and animal RNA viruses. Direct sequence analysis of the RhPV capsid proteins showed that ORF2, which represents the last 2900 nt, encodes the three structural proteins (28, 29, and 30 kDa). The predicted amino acid sequence of ORF2 is very similar to the corresponding regions ofDrosophilaC virus,Plautia staliintestine virus, and to a partial sequence from the 3′ end of the cricket paralysis virus genome. The site of initiation of protein synthesis for ORF2 could not be determined from the amino acid and nucleotide sequences. ORF1 is preceded by 579 nt of noncoding RNA and the two ORFs are separated by more than 500 nt of noncoding RNA. Like picornaviruses, these regions may function to facilitate the cap-independent initiation of translation of the two ORFs. These data suggest that RhPV,DrosophilaC virus,Plautia staliintestine virus, and probably cricket paralysis virus are members of a unique group of small RNA viruses that infect primarily insects.

Cloning and nucleotide sequence of the nitric oxide reductase locus in Paracoccus denitrificans IFO 12442

A 5.2-kb DNA fragment containing the upstream region of the nitrite reductase gene ( nirS) was cloned from Paracoccus denitrificans IFO 12442 and its DNA sequence was determined. In this fragment, four open reading frames (ORFs) were observed. Among these, two ORFs, located 3 kb upstream of the nirS gene were found to encode nitric oxide reductase ( norC, norB) by homology analysis. The norC and norB encoded cytochrome c and b subunits of the enzyme, and the predicted molecular weights of the protein products were 17 kDa (150 amino acid residues) and 52.5 kDa (462 amino acid residues), respectively. The other two open reading frames, designated ORF1 (73 kDa, 681 amino acid residues) and ORF2 (32.5 kDa, 304 amino acid residues), were found between the nir and nor genes. The deduced amino acid sequence of ORF1 showed high similarity (35% identity) to that of NosR which is known to be a transcriptional regulatory protein for the N 2O reductase gene cluster of Pseudomonas stutzeri. Since the ORF1 protein has a well conserved cysteine cluster and 5 hydrophobic transmembrane repeats similar to those of the NosR protein, ORF1 probably functions as a membrane bound sensor for denitrification. Northern blot analysis indicated that the ORF1–2 and norCB regions are probably transcribed as independent operons.

Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor.

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.