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PubMed1992

Lactate dehydrogenase-elevating virus (LDV): subgenomic mRNAs, mRNA leader and comparison of 3'-terminal sequences of two LDV isolates.

The 3'-terminal 1314 nucleotides of the genome of one isolate of lactate dehydrogenase-elevating virus, LDV-P, has been derived by sequence analyses of cDNAs from several genomic libraries and compared to that of another LDV isolate, LDV-C (Godeny et al. (1990) Virol. 177, 768-771). The 3'-non-coding segment of 80 nucleotides of the two LDV genomes is identical, whereas marked, but varying nucleotide and amino acid divergence is apparent in the three upstream overlapping open reading frames (ORF). The third ORF from the 3'-end exhibits only 82% nucleotide and 90% amino acid identity, whereas the 3'-terminal ORF, which encodes the nucleocapsid protein, exhibits approximately 99% amino acid identity. The second 3'-terminal ORF encodes an 18.8 kDa protein which lacks N-glycosylation sites but possesses 2 or 3 potential transmembrane helices in the N-terminal half of the molecule. A similar membrane organization is observed for the corresponding protein of equine arteritis virus and the M protein of mouse hepatitis virus. The sequence analyses combined with Northern hybridization analyses of RNA from LDV-infected macrophages and spleens of LDV-infected mice indicate that the three ORFs encoded by the 3'-terminal end of the LDV genome are expressed via the three smallest mRNAs (mRNAs 6-8) of the seven subgenomic mRNAs of LDV (mRNAs 2-8), which range in size from about 0.8 to 3.6 kb. All mRNAs have been shown to carry poly(A)-tracts and a common leader sequence. The seven mRNAs were produced in infected macrophage cultures concomitantly with genomic LDV RNA. Maximum LDV RNA synthesis was observed between 6 and 8 h post-infection. The same seven subgenomic mRNAs were detected in macrophages infected with three different isolates of LDV, but different relative amounts of some of the mRNAs were produced. The relative proportions of molecules of mRNAs 1-8 present in 6 h LDV-P-infected macrophages were about 13, 5, 5, 8, 6, 11, 11 and 27% of the total, respectively.

Amino Acid Sequence↗

PubMed1986

Construction of LYS2 cartridges for use in genetic manipulations of Saccharomyces cerevisiae.

Linker arrays were added to the 5' and 3' boundaries of the Saccharomyces cerevisiae LYS2 gene, which allow the generation of 18 LYS2 cartridges with different sticky ends. As it was necessary to define the beginning and the end of the approx. 4.5-kb LYS2 gene, we sequenced 1 kb of its 5' and 1.5 kb of its 3' region and mapped the mRNA start point. The open reading frame (ORF) found by this analysis was proven to be the LYS2 ORF by exchanging the sequences upstream from the presumptive ATG with the S. cerevisiae CYC1 promoter and subsequent demonstration of LYS2 expression in vivo. The proper functioning of the LYS2 cartridges was demonstrated by the transformation of lys2 mutant strains to Lys+ prototrophy using plasmids furnished with a LYS2 cartridge.

Aldehyde Oxidoreductases↗

PubMed2002

Genome organization of the linear Pichia etchellsii plasmid pPE1A: evidence for expression of an extracellular chitin-binding protein homologous to the alpha-subunit of the Kluyveromyces lactis killer toxin.

Pichia etchellsii CBS2011 (synonym Debaryomyces etchellsii) is a non-killer yeast harbouring two cryptic linear cytoplasmic DNA-elements, pPE1A (6.7 kb) and pPE1B (12.8 kb). Cloning and complete sequencing of pPE1A revealed a 6749-bp element with a remarkably high A+T content of 77.6%. The termini of pPE1A were found to consist of inversely orientated identical nucleotide repetitions of 178bp, to which proteins are linked at the 5'-ends. It is only the second small, non-autonomous cytoplasmic yeast linear plasmid for which the complete nucleotide sequence is known. Five open reading frames (ORFs) were identified preceded by upstream conserved sequence motifs (UCS) characteristic for cytoplasmic promoters and perfectly matching the UCS consensus (ATNTGA). As none of the putative genes encodes a DNA-polymerase, pPE1A is the first yeast linear plasmid known that does not possess its own element-specific replication machinery. No function could be attributed to ORF1, 3, 4, and 5; the predicted ORF2 gene product is similar to chitin-binding proteins and chitinases, highest homologies were found to the precursor of the alpha- and beta-subunits of the secreted Kluyveromyces lactis zymocin. Consistently, the Orf2p could be isolated from the culture fluid by chitin-Sepharose affinity chromatography and characterized by immuno-probing with an antibody specific for the K. lactis killer toxin alpha-subunit. Production of the protein was found to be plasmid-dependent. The sequence of pPE1A has been submitted to the EMBL data library, Accession No. AJ409097.

Amino Acid Sequence↗

PubMed1997

Sequence of a melibiose transporter gene of Enterobacter cloacae.

We cloned a fragment of the chromosomal DNA of Enterobacter cloacae, which enabled a melibiose-negative Escherichia coli mutant lacking melB to grow on melibiose as the sole source of carbon. Transformed cells harboring the hybrid plasmid carrying the cloned DNA showed melibiose transport activity. The nucleotide sequence of the DNA region was determined. One complete open reading frame (ORF) and a part of another ORF were found in the region, and the amino acid sequences were deduced. The complete ORF was found to encode a melibiose transporter which consisted of 425 amino acid residues. Hydropathy analysis revealed that there are about 12 hydrophobic domains in this transporter. The incomplete ORF which exists in the upstream region of the transporter gene seemed to encode an alpha-galactosidase.

Amino Acid Sequence↗

PubMed1998

Evidence for a bidirectional promoter complex within the X gene of woodchuck hepatitis virus.

The genetic organization of hepadnaviruses is unusual in that all cis-acting regulatory sequences are located within genes. Thus, in the mammalian hepadnavirus genome, the presurface, surface, and X transcript promoters reside within the polymerase gene while the pregenome transcript promoter is located within the X gene. In this study we have identified two additional promoters within the woodchuck hepatitis virus (WHV) X gene that stimulate production of transcripts in vitro. First, we cloned regions of the WHV X gene into a promoterless expression vector (pGL2) to examine their ability to promote expression of firefly luciferase and mapped a previously unidentified promoter to positions 1475-1625 of the WHV8 genome. Deletion analysis revealed that the essential domain of this promoter, termed the ORF5/deltaX transcript promoter, mapped to nucleotides 1525-1625. Analysis revealed that this transcript initiated at nucleotide 1572 in both human (HuH-7) and woodchuck (WLC-3) hepatoma cell lines. Consistent with this finding, DNA footprinting analysis revealed protection of nucleotides 1567-1578 on the positive strand of the WHV8 genome. The function of this transcript in vivo is unclear, however, it may be used to produce a truncated form of the X protein that initiates at an AUG codon at position 1743-1745 on the WHV8 genome. Next, a second promoter was identified at positions 1625-1975 that was responsible for production of an antisense transcript. The activity of this promoter was comparable to that of the previously characterized surface transcript promoter of WHV in the absence of an enhancer. The antisense transcript promoter resides immediately upstream of open reading frame (ORF) 6, a previously identified ORF on the strand opposite of the known WHV protein-encoding sequences, that is thought to represent a vestigial gene. Analysis indicates that the antisense transcript had multiple start sites: nucleotides 1683 and 1762 on the WHV8 genome when assayed in HuH-7 cells, and nucleotide 1786 when assayed in WLC-3 cells. These data are consistent with footprinting analysis of supercoiled WHV DNA that revealed that the regions encompassing nucleotides 1696-1685, 1781-1766, and 1801-1787 on the negative sense DNA strand were protected from nuclease degradation. It is possible that such a transcript was once used in protein expression in an ancestral virus and may now be used for genetic control of WHV replication and/or gene expression. Overall, these data are consistent with the presence of a bidirectional promoter complex within the WHV X gene.

Animals↗

PubMed1997

A ColE1-type plasmid from Salmonella enteritidis encodes a DNA cytosine methyltransferase.

The multicopy plasmid pFM366 was isolated from a virulent Salmonella enteritidis strain and was found to code for DNA methylase activity (Ibáñez and Rotger, 1993). The present work was aimed at characterizing the genetic organization and functional features of this 5.6 kb plasmid. We found pFM366 almost identical to the plasmid P4 isolated from Shigella sonnei, that encodes the SsoII restriction-modification system (Karyagina et al., 1993), and related to other ColE1-type plasmids. Examination of these plasmids revealed a common organization which suggests they were the result of similar recombinational events. The cytosine methylase of pFM366 is nearly identical to M. SsoII, whereas the gene encoding the restrictase homologous to R. SsoII is truncated and its product is inactive. The expression of the cytosine methylase encoded by pFM366 is strongly affected by deletion of regions located upstream and downstream of its ORF, and is negatively controlled by the rpoS gene in Escherichia coli. The methylase activity encoded by pFM366 induces the SOS response, which could be responsible for the observed delay in the growth of E. coli.

Amino Acid Sequence↗

PubMed2000

Comparative analysis of the granulin regions of the Phthorimaea operculella and Spodoptera littoralis granuloviruses.

The nucleotide sequence of two cloned restriction fragments encompassing the granulin genes from the granuloviruses of the potato tuber moth, Phthorimaea operculella, PhopGV, and the Egyptian cotton leaf worm, Spodoptera littoralis, SpliGV, have been determined. Although both viruses are able to infect the same Ph. operculella cell line, their granulins do not cluster in the same phylogenetic branches. PhopGV ganulin is closely related to Cydia pomonella GV (CpGV) and Cryptophlebia leucotreta GV (ClGV) (95.2 and 94% identity at the aminoacid level), while SpliGV granulin falls close to Trichoplusia ni GV and Xestia c-nigrum GV (91.6 and 92.0% respectively). The gene organization around the granulins reflects this clustering. Upstream the PhopGV granulin, an ORF belonging to the ME53 gene family (as ORF 124R of CpGV and 909 of ClGV) is present, while no equivalent ORF is found in this region in SpliGV. Downstream the granulin, both viruses present a gene homologous to the Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 9 followed by a Protein Kinase (AcMNPV ORF10). The structure of this region seems thus conserved not only among nucleopolyhedroviruses but also in at least some granuloviruses.

Animals↗

PubMed1999

Biochemical and molecular characterization of the [NiFe] hydrogenase from the hyperthermophilic archaeon, Thermococcus litoralis.

Thermococcus litoralis is a hyperthermophilic archaeon that grows at temperatures up to 98 degrees C by fermentative metabolism and reduces elemental sulfur (S0) to H2S. A [NiFe] hydrogenase, responsible for H2S or H2 production, has been purified and characterized. The enzyme is composed of four subunits with molecular mass 46, 42, 34 and 32 kDa. Elemental analyses gave approximate values of 22 Fe, 22 S and 1 Ni per hydrogenase. EPR spectra at 70 and 5 K indicated the presence of four or five [4Fe-4S] and one [2Fe-2S] type clusters. The optimal temperature for both H2 evolution and oxidation, using artificial electron carriers, was around 80 degrees C. The operon encoding the T. litoralis enzyme is composed of four genes forming one transcriptional unit, and transcription is not regulated by S0. An unusual transcription-initiation site is located 139 bp upstream from the translational start point. Sequence analyses indicated the presence of new putative nucleotide-binding domains. Upstream from the hydrogenase operon, ORFs probably encoding a molybdopterin oxidoreductase enzyme have been identified. Based on sequence, biochemical and biophysical analyses, a model of the enzyme and the pathway of electron flow during catalysis is proposed.

Amino Acid Sequence↗

PubMed1997

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea.

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5' upstream region of the grisea ORF. One mutation is a G --> A transition at the 5' splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

Ascomycota↗

PubMed1999

North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions.

Although North American and European serotypes of porcine reproductive and respiratory syndrome virus (PRRSV) are recognized, only the genome of the European Lelystad strain (LV) has been sequenced completely. Here, the genome of the pathogenic North American PRRSV isolate 16244B has been sequenced and compared with LV. The genomic organization of 16244B was the same as LV but with only 63.4% nucleotide identity. The 189 nucleotide 5' non-coding region (NCR) of 16244B was distinct from the LV NCR, with good conservation (83%) only over a 43 base region immediately upstream of open reading frame (ORF) 1a. Major differences were found in the region encoding the non-structural part of the ORF1a polyprotein, which shared only 47% amino acid identity over 2503 residues of the six non-structural proteins (Nsps) encoded. Nsp2, thought to have a species-specific function, showed the greatest divergence, sharing only 32% amino acid identity with LV and containing 120 additional amino acids in the central region. Nsps encoded by the 5'-proximal and central regions of ORF1b had from 66 to 75% amino acid identity; however, the carboxy-terminal protein CP4 was distinct (42% identity). The ORF 1a-1b frameshift region of 16244B had 98% nucleotide identity with LV. Consistent with previous reports for North American isolates, the six structural proteins encoded were 58 to 79% identical to LV proteins. The 3' NCR (150 nucleotides) was 76% identical between isolates. These genomic differences confirm the presence of distinct North American and European PRRSV genotypes.

5' Untranslated Regions↗

PubMed1997

Genome and MIC stability in Mycobacterium tuberculosis and indications for continuation of use of isoniazid in multidrug-resistant tuberculosis.

Mycobacterium tuberculosis strains resistant to two or more of the first line antituberculosis drugs (MDR) are a serious threat to successful tuberculosis control programmes. For this retrospective study, 85 follow-up drug resistant isolates from 23 patients residing in a community with a high incidence of tuberculosis were collected and the level of in-vitro resistance to antibiotics determined quantitatively. PCR-SSCP and sequencing techniques were used to screen for gene mutations associated with resistance in 31 follow-up samples from a smaller group of eight patients. DNA fingerprint analysis was done on sequential isolates to confirm identity. Although treatment had a profound effect on changes in drug resistance patterns, the MIC for a particular agent remained constant in follow-up isolates. DNA fingerprinting and mutational analysis (14 different loci) showed that the genome of MDR strains of M. tuberculosis is relatively stable during the course of therapy. The rpoB gene was the most frequently mutated structural gene involved in drug resistance and a novel C to T mutation upstream of open reading frame (ORF)1 of the inhA operon was detected. No evidence was found of the presence of strain W (New York) in this group of MDR strains. The results stress the importance of confirming individuality of strains for the accurate calculation of frequencies of particular mutations associated with drug resistance, particularly in a high incidence area. Approximately one-half (47.8%) of the patients had isolates resistant to concentrations just above the critical concentration for isoniazid (MICs of 0.2-5 mg/L). Therefore, these patients and their contacts who develop primary drug-resistant tuberculosis may respond to higher dosages of treatment which could have a considerable impact on the cost and the ease of management of resistant tuberculosis.

Antitubercular Agents↗

PubMed1994

Cloning and DNA sequence analysis of the region containing attP of the temperate phage phi AR29 of Prevotella ruminicola AR29.

Phage phi AR29 was shown to exist as a prophage integrated into the chromosome of Prevotella ruminicola AR29. By DNA hybridization studies, the point of integrative recombination on the phage genome (attP) was located on a 4.5 kb EcoRV fragment. After preliminary mapping with restriction endonucleases, a 2.8 kb EcoRV/HindIII fragment was isolated, cloned in Escherichia coli and sequenced. DNA hybridization localized the attP site to the vicinity of an internal DraI site. Sequence analysis showed the presence of several direct and inverted repeats around the attP site, with consensus core sequences similar to the integrase binding sites of phage lambda. Two open reading frames are present adjacent to attP (ORF1 and ORF2). The predicted polypeptide product of ORF1 has a region of structural similarity to known integrases. Although the predicted product of ORF2 shows at best weak homology with known excisionases, no other ORFs occur in the sequence upstream from ORF1, leaving ORF2 as the most likely candidate for this role. However, if ORF2 does represent an xis gene, then this putative integration module would possess a notable difference from that of other temperate phages in the inversion of the positions of int and xis relative to attP. The proposed phi AR29 integration module is being used to develop phage-based integrative vector systems for the genetic manipulation of rumen bacteria.

Amino Acid Sequence↗

PubMed2005

bph genes of the thermophilic PCB degrader, Bacillus sp. JF8: characterization of the divergent ring-hydroxylating dioxygenase and hydrolase genes upstream of the Mn-dependent BphC.

Bacillus sp. JF8 is a thermophilic polychlorinated biphenyl (PCB) degrader, which utilizes biphenyl and naphthalene. A thermostable, Mn-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase, BphC_JF8, has been characterized previously. Upstream of bphC are five ORFs exhibiting low homology with, and a different gene order from, previously characterized bph genes. From the 5' to 3' direction the genes are: a putative regulatory gene (bphR), a hydrolase (bphD), the large and small subunits of a ring-hydroxylating dioxygenase (bphA1A2), and a cis-diol dehydrogenase (bphB). Hybridization studies indicate that the genes are located on a plasmid. Ring-hydroxylating activity of recombinant BphA1A2_JF8 towards biphenyl, PCB, naphthalene and benzene was observed in Escherichia coli cells, with complementation of non-specific ferredoxin and ferredoxin reductase by host cell proteins. PCB degradation by recombinant BphA1A2_JF8 showed that the congener specificity of the recombinant enzyme was similar to Bacillus sp. JF8. BphD_JF8, with an optimum temperature of 85 degrees C, exhibited a narrow substrate preference for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid. The Arrhenius plot of BphD_JF8 was biphasic, with two characteristic energies of activation and a break point at 47 degrees C.

Bacillus↗

PubMed1997

Analysis of the icsBA locus required for biosynthesis of the inner core region from Neisseria meningitidis lipopolysaccharide.

By deletion mutagenesis in the entire meningococcal chromosome, we have previously identified the icsA gene, which encodes the glycosyltransferase required for adding GlcNAc to Hep-II in the inner core of meningococcal LPS. This gene has homology to several LPS glycosyltransferases, notably to rfaK from Salmonella typhimurium and bplH from Bordetella pertussis, both of which encode GlcNAc transferases. Directly upstream of icsA is an ORF showing significant homology to the hypothetical protein HI0653 from the Haemophilus influenzae genome sequence, and to a lesser degree to putative glycosyltransferases from Streptococcus thermophilus and Yersinia enterocolitica. Insertional inactivation of this ORF resulted in a meningococcal strain with truncated LPS. We have named this new LPS-involved gene icsB. Differences in binding of monoclonal antibodies and in mobility on Tricine-SDS-PAGE showed that LPS from icsA and icsB mutants is similar but not identical. On the basis of these results, we postulated that the new gene encodes the glycosyltransferase required for adding Glc to Hep-I. Structural analysis of purified mutant LPS by electrospray mass spectrometry was used to verify this hypothesis. The composition determined for icsA and icsB is lipidA-(KDO)2-(Hep)2.PEA and lipidA-(KDO)2-(Hep)2.PEA-GlcNAc, respectively. The icsA and icsB genes thus form an operon encoding the glycosyltransferases required for chain elongation from the lipidA-(KDO)2-(Hep)2 basal structure, with IcsA first adding GlcNAc to Hep-II and IcsB subsequently adding Glc to Hep-I. Only then is completion of the lacto-N-neotetraose structure possible through the action of the IgtA-E genes.

Acetylglucosamine↗

PubMed1994

Characterization of the gene encoding hydroxylamine oxidoreductase in Nitrosomonas europaea.

Hydroxylamine oxidoreductase (HAO) catalyzes the oxidation of hydroxylamine to nitrite in Nitrosomonas europaea. The electrons released in the reaction are partitioned to ammonium monooxygenase and to the respiratory chain. The immediate acceptor of electrons from HAO is believed to be cytochrome c-554 (Cyt c-554). We have isolated a genomic DNA fragment containing the structural gene encoding HAO (hao) and a part of the gene for Cyt c-554. The nucleotide sequence of hao was determined, and its transcription was analyzed. The open reading frame (ORF) encodes amino acid sequences matching the purified peptides of HAO. A 64.28-kDa protein is encoded in this ORF, in close agreement with the empirically determined molecular mass of 63 kDa. The N terminus was located 24 amino acids from the start codon, suggesting the presence of a leader sequence. The putative eight heme-binding peptides were localized in this ORF. The gene for Cyt c-554 was located 1,200 bp downstream from the 3' end of hao. An ORF was identified in the upstream region from hao and may encode a protein of unknown function. Data bank searches did not reveal proteins with substantial similarities to HAO, but they did reveal similarities between Cyt c-554 and other c-type cytochromes.

Amino Acid Sequence↗

PubMed1998

Sendai virus Y proteins are initiated by a ribosomal shunt.

The Sendai virus P/C mRNA expresses eight primary translation products by using a combination of ribosomal choice and cotranscriptional mRNA editing. The longest open reading frame (ORF) of the mRNA starts at AUG104 (the second initiation site) and encodes the 568-amino-acid P protein, an essential subunit of the viral polymerase. The first (ACG81), third (ATG114), fourth (ATG183), and fifth (ATG201) initiation sites are used to express a C-terminal nested set of polypeptides (collectively named the C proteins) in the +1 ORF relative to P, namely, C', C, Y1, and Y2, respectively. Leaky scanning accounts for translational initiation at the first three start sites (a non-ATG followed by ATGs in progressively stronger contexts). Consistent with this, changing ACG81/C' to ATG (GCCATG81G) abrogates expression from the downstream ATG104/P and ATG114/C initiation codons. However, expression of the Y1 and Y2 proteins remains normal in this background. We now have evidence that initiation from ATG183/Y1 and ATG201/Y2 takes place via a ribosomal shunt or discontinuous scanning. Scanning complexes appear to assemble at the 5' cap and then scan ca. 50 nucleotides (nt) of the 5' untranslated region before being translocated to an acceptor site at or close to the Y initiation codons. No specific donor site sequences are required, and translation of the Y proteins continues even when their start codons are changed to ACG. Curiously, ATG codons (in good contexts) in the P ORF, placed either 16 nt upstream of Y1, 29 nt downstream of Y2, or between the Y1 and Y2 codons, are not expressed even in the ACGY1/ACGY2 background. This indicates that ATG183/Y1 and ATG201/Y2 are privileged start sites within the acceptor site. Our observations suggest that the shunt delivers the scanning complex directly to the Y start codons.

Base Sequence↗

PubMed1986

Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product.

We previously described use of the human parvovirus, adeno-associated virus (AAV), as a vector for transient expression in mammalian cells of the gene for chloramphenicol acetyltransferase (CAT). In the AAV vector, pTS1, the CAT gene is expressed under the control of the major AAV promoter p40. This promoter is embedded within the carboxyl-terminal region of an open reading frame (orf-1) which codes for a protein (rep) required for AAV DNA replication. We show here that the rep product has additional trans-acting properties to regulate gene expression. First, deletion or frame-shift mutations in orf-1, which occurred far upstream of p40, increased expression of CAT in human 293 (adenovirus-transformed) cells. This increased CAT expression was abolished when such mutant AAV vectors were transfected into 293 cells together with a second AAV vector which could supply the wild-type AAV rep product in trans. Thus, an AAV rep gene product was a negative regulator, in trans, of expression of CAT in uninfected 293 cells. In adenovirus-infected 293 cells, the function of the AAV rep product was more complex, but in some cases, it appeared to be a trans activator of the expression from p40. In HeLa cells, only trans activation by rep was seen in the absence or presence of adenovirus. Neither activation nor repression by the rep product required replication per se of the AAV vector DNA. Thus, trans-acting negative or positive regulation of gene expression by the AAV rep gene is modulated by factors in the host cell and by the helper adenovirus.

Acetyltransferases↗

PubMed2000

Cloning and characterization of the iutA gene which encodes ferric aerobactin receptor from marine Vibrio species.

The iutA gene from marine Vibrio species SD004, which encoded a ferric aerobactin receptor for the uptake of iron(III), was cloned onto a multicopy plasmid, pUC 18, in Escherichia coli. Identification of the positive clone was achieved on the basis of its deferrization activity and was detected as a halo formation on the chrome azurol S (CAS)-containing selective plate. Nucleotide sequence analysis of the cloned DNA fragment revealed an open reading frame (ORF) which encoded a polypeptide of 706 amino acid residues, and the deduced molecular mass of this polypeptide was 77.906 kD. The amino acid sequence showed a 41% homology with that of the lutA protein from E. coli. The cloned gene was iutA, which encoded the ferric aerobactin receptor. Another incomplete ORF was found 100 bp upstream of the iutA gene, which was homologous (31 out of 49 amino acids) with the C-terminal region of the luc D protein of E. coli. It is suggested that aerobactin biosynthesis and the transport genes are located tandemly on the Vibrio chromosome and may form an aerobactin operon.

Amino Acid Sequence↗