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S K Farrand

Publications and source records attributed to S K Farrand.

At least 37 records · Page 2Linked to original sources
PubMed1999

Conjugal transfer but not quorum-dependent tra gene induction of pTiC58 requires a solid surface.

Donors of Agrobacterium tumefaciens harboring a transfer-constitutive derivative of the nopaline-type Ti plasmid pTiC58 transferred this element at frequencies 3 to 4 orders of magnitude higher in matings conducted on solid surfaces than in those conducted in liquid medium. However, as measured with a lacZ reporter fusion, the tra genes of the wild-type Ti plasmid were inducible by opines to indistinguishable levels on solid and in liquid medium. Donors induced in liquid transferred the Ti plasmid at high frequency when mated with recipients on solid medium. We conclude that while formation of stable mating pairs and subsequent transfer of the Ti plasmid is dependent on a solid stratum, the regulatory system can activate tra gene expression to equivalent levels in liquid and on solid surfaces.

Agrobacterium tumefaciens↗
PubMed1999

Essential components of the Ti plasmid trb system, a type IV macromolecular transporter.

The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production of Agrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58DeltaaccR mutations in trbB, -C, -D, -E, -L, -F, -G, and -H abolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation in traI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of the trb genes except traI and trbJ can be complemented by functions coded for by pAtC58.

Agrobacterium tumefaciens↗
PubMed1999

Cloning and characterization of a tetracycline resistance determinant present in Agrobacterium tumefaciens C58.

Agrobacterium tumefaciens C58 and its derivatives give rise to spontaneous mutants resistant to tetracycline at a high frequency. We observed that a mutation affecting a tRNA processing function significantly affected the emergence of such mutants, suggesting that C58 contained a positively acting gene conferring resistance to tetracycline. A cosmid clone conferring resistance to tetracycline in Escherichia coli and Agrobacterium was isolated from a genomic bank of one such mutant. Subcloning, transposon mutagenesis, and DNA sequence analysis revealed that this DNA fragment contained two divergently transcribed genes, tetA and tetR, encoding products that were very similar to proteins of the Tet(A) class of tetracycline resistance systems. In the clone from this mutant, tetR was disrupted by an IS426. The homologous region from wild-type NT1 contained an intact tetR gene and did not confer resistance to tetracycline. Hybridization analysis showed that of 22 members of the genus Agrobacterium surveyed, only strains C58 and T37 contained the tet determinant. Moreover, only these two strains mutated to resistance to this antibiotic. Unlike other Tet(A) systems, neither tetracycline nor a series of its derivatives induced the expression of this tet gene unit. Other polycyclic compounds, including many of plant origin, also did not induce this tet gene system. The divergent promoter region of this tet system contained a single inverted repeat element identical to one such operator repeat in the promoter region of the tet determinant from the IncP1alpha R plasmid RP4. TetR repressor proteins from the Agrobacterium tet system and from RP4 interacted with the heterologous operators. While the repressive effect of the TetR protein from strain C58 (TetRC58) on the tetA gene from strain RP4 (tetARP4) was not relieved by tetracycline, repression of tetAC58 by TetRRP4 was lifted by this antibiotic.

Agrobacterium tumefaciens↗
PubMed1998

Octopine-type Ti plasmids code for a mannopine-inducible dominant-negative allele of traR, the quorum-sensing activator that regulates Ti plasmid conjugal transfer.

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by two hierarchical signalling systems. Transfer is dependent on a subset of opines produced by the plant tumours induced by the bacterium. Induction also requires an acyl-homoserine lactone signal, called AAI, that is produced by the bacteria themselves. AAI is the co-inducer for TraR, the transcriptional activator required for expression of the tra regulon. Octopine induces conjugation of the octopine-mannityl opine-type Ti plasmids by regulating the expression of traR via OccR, the octopine-dependent activator of the opine regulon. We have discovered a second traR-like gene, trlR, on the octopine-mannityl opine-type Ti plasmids pTi15955 and pTiR10. This gene is located in an operon coding for a mannopine transport system and is expressed as part of the mannityl opine regulon. Sequence analysis indicated that trlR is a frameshift allele of traR, and the resulting protein lacks the carboxy-terminal domain thought to constitute the DNA-binding region of TraR. Expression of trlR inhibited octopine-induced conjugation of pTi15955 and pTiR10 by suppressing the TraR-mediated transcription of the tra and trb operons. Although TrlR had no effect on the expression of traR, TraR activated the expression of trlR. Southern hybridizations indicated that several other Ti and opine-catabolic plasmids contain more than one copy of genes homologous to traR. We propose that trlR is a dominant negative allele of traR and that TrlR inhibits conjugation by forming inactive heteromultimers with TraR.

Agrobacterium tumefaciens↗
PubMed1998

Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria.

Many gram-negative bacteria regulate expression of specialized gene sets in response to population density. This regulatory mechanism, called autoinduction or quorum-sensing, is based on the production by the bacteria of a small, diffusible signal molecule called the autoinducer. In the most well-studied systems the autoinducers are N-acylated derivatives of L-homoserine lactone (acyl-HSL). Signal specificity is conferred by the length, and the nature of the substitution at C-3, of the acyl side-chain. We evaluated four acyl-HSL bioreporters, based on tra of Agrobacterium tumefaciens, lux of Vibrio fischeri, las of Pseudomonas aeruginosa, and pigment production by Chromobacterium violaceum, for their ability to detect sets of 3-oxo acyl-HSLs, 3-hydroxy acyl-HSLs, and alkanoyl-HSLs with chain lengths ranging from C4 to C12. The traG::lacZ fusion reporter from the A. tumefaciens Ti plasmid was the single most sensitive and versatile detector of the four. Using this reporter, we screened 106 isolates representing seven genera of bacteria that associate with plants. Most of the Agrobacterium, Rhizobium, and Pantoea isolates and about half of the Erwinia and Pseudomonas isolates gave positive reactions. Only a few isolates of Xanthomonas produced a detectable signal. We characterized the acyl-HSLs produced by a subset of the isolates by thin-layer chromatography. Among the pseudomonads and erwinias, most produced a single dominant activity chromatographing with the properties of N-(3-oxo-hexanoyl)-L-HSL. However, a few of the erwinias, and the P. fluorescens and Ralstonia solanacearum isolates, produced quite different signals, including 3-hydroxy forms, as well as active compounds that chromatographed with properties unlike any of our standards. The few positive xanthomonas, and almost all of the agrobacteria, produced small amounts of a compound with the chromatographic properties of N-(3-oxo-octanoyl)-L-HSL. Members of the genus Rhizobium showed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals. Several isolates produced extremely nonpolar compounds indicative of very long acyl side-chains. Production of these compounds suggests that quorum-sensing is common as a gene regulatory mechanism among gram-negative plant-associated bacteria.

4-Butyrolactone↗
PubMed1998

Opine catabolic loci from Agrobacterium plasmids confer chemotaxis to their cognate substrates.

Opines are carbon compounds produced by crown galls and hairy roots induced by Agrobacterium tumefaciens and A. rhizogenes, respectively. These novel condensation products of plant metabolic intermediates are utilized as nutritional sources by the Agrobacterium strains that induced the growths. Thus, opines are thought to favor the propagation of agrobacteria in the tumorsphere. Certain Agrobacterium strains were chemoattracted to opines. The chemotactic activities to octopine, to nopaline, to mannopine, and to agrocinopines A + B were dependent on the type of the Ti plasmid present in the bacterium. The determinants for chemotaxis to these opines were localized to the regions of the octopine- and nopaline-type Ti plasmids coding for transport and catabolism of that opine. An insertion in accA, which encodes the periplasmic binding protein for agrocinopines A + B, abolished chemotaxis while an insertion in accC, which encodes a component of the transport system, and an insertion in accF, which encodes a function required for agrocinopine catabolism, did not affect chemotaxis to this opine. Thus, transport and catabolism of these opines are not required for the chemotactic activity. Analyses of subclones of the acc region confirmed that accA is the only gene required from the Ti plasmid for chemotaxis to agrocinopines A + B.

Carbon↗
PubMed1998

Genetic and sequence analysis of the pTiC58 trb locus, encoding a mating-pair formation system related to members of the type IV secretion family.

Conjugal transfer of pTiC58 requires two regions, tra which contains the oriT and several genes involved in DNA processing and a region of undefined size and function that is located at the 2-o'clock position of the plasmid. Using transposon mutagenesis with Tn3HoHo1 and a binary transfer system, we delimited this second region, called trb, to an 11-kb interval between the loci for vegetative replication and nopaline catabolism. DNA sequence analysis of this region identified 13 significant open reading frames (ORFs) spanning 11,003 bp. The first, encoding traI, already has been described and is responsible for the synthesis of Agrobacterium autoinducer (AAI) (I. Hwang, P.-L. Li, L. Zhang, K. R. Piper, D. M. Cook, M. E. Tate, and S. K. Farrand, Proc. Natl. Acad. Sci. USA 91:4639-4643, 1994). Translation products of the next 11 ORFs showed similarities to those of trbB, -C, -D, -E, -J, -K, -L, -F, -G, -H, and -I of the trb region of the octopine-type Ti plasmid pTi15955 and of the tra2 core region of RP4. In RP4, these genes encode mating-pair formation functions and are essential for the conjugal transfer of the IncP plasmid. Each of the trb gene homologues is oriented counterclockwise on the Ti plasmid. Expression of these genes, as measured by using the lacZ fusions formed by Tn3HoHo1, required the traI promoter and the transcriptional activator TraR along with its coinducer, AAI. While related to that of RP4, the trb system of pTiC58 did not allow propagation of the trb-specific bacteriophages PRD1, PRR1, and Pf3. The products of several trb genes of the Ti plasmid are similar to those of other loci that encode DNA transfer or protein secretion systems, all of which are members of the type IV secretion family.

Agrobacterium tumefaciens↗
PubMed1997

Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography.

Many Gram-negative bacteria regulate gene expression in response to their population size by sensing the level of acyl-homoserine lactone signal molecules which they produce and liberate to the environment. We have developed an assay for these signals that couples separation by thin-layer chromatography with detection using Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. With the exception of N-butanoyl-L-homoserine lactone, the reporter detected acyl-homoserine lactones with 3-oxo-, 3-hydroxy-, and 3-unsubstituted side chains of all lengths tested. The intensity of the response was proportional to the amount of the signal molecule chromatographed. Each of the 3-oxo- and the 3-unsubstituted derivatives migrated with a unique mobility. Using the assay, we showed that some bacteria produce as many as five detectable signal molecules. Structures could be assigned tentatively on the basis of mobility and spot shape. The dominant species produced by Pseudomonas syringae pv. tabaci chromatographed with the properties of N-(3-oxohexanoyl)-L-homoserine lactone, a structure that was confirmed by mass spectrometry. An isolate of Pseudomonas fluorescens produced five detectable species, three of which had novel chromatographic properties. These were identified as the 3-hydroxy- forms of N-hexanoyl-, N-octanoyl-, and N-decanoyl-L-homoserine lactone. The assay can be used to screen cultures of bacteria for acyl-homoserine lactones, for quantifying the amounts of these molecules produced, and as an analytical and preparative aid in determining the structures of these signal molecules.

4-Butyrolactone↗
PubMed1997

Modification of rhizobacterial populations by engineering bacterium utilization of a novel plant-produced resource.

The ability to catabolize distinct nutrients produced by a plant may be a factor in the successful colonization of that host by a bacterium when in competition with other rhizosphere microorganisms. We tested this hypothesis by examining the influence of a novel substrate produced by a transgenic plant on root colonization by near-isogenic bacteria, differing only in their ability to use the resource. When inoculated alone, both bacteria colonized the roots of the normal and transgenic plants with equal kinetics and to indistinguishable levels. When the two bacteria were coinoculated, the catabolizer reached a population density significantly higher than that of the noncatabolizer on the roots of the resource-producing plant. No such advantage was observed on the roots of normal plants. These results support the theory that resources produced and exuded by a plant host can confer a selective advantage to microorganisms that use the substrate.

Agrobacterium tumefaciens↗
PubMed1997

Detection and Enumeration of a Tagged Pseudomonas fluorescens Strain by Using Soil with Markers Associated with an Engineered Catabolic Pathway.

Previously we described a novel gene tagging method, using the moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955, to identify microorganisms destined for release into the environment. Here, we used the engineered strain Pseudomonas fluorescens PF5MT12 carrying the moc region integrated into the bacterial chromosome to demonstrate the usefulness of the markers for detection and direct selection of marked organisms present in soil samples. Using this system, we routinely detected population levels as low as 10(sup2) CFU per g of soil sampled. In addition to direct selection, we developed an immunologically based assay using MOP cyclase, a unique enzyme associated with moc, as the epitope for detecting the tagged organism. The colony immunoblot assay proved to be highly specific and without any false-positive signals when used to identify organisms cultured from soil on nonselective medium. The numbers of colonies that were immunoreactive with the anti-MOP cyclase antibody were essentially equal to those that grew out on selection plates. This indicates that MOP cyclase can be used as a marker and that we can use nonselective medium to retrieve the marked genetically engineered microorganisms and then identify them by using colony immunoblot assays. These direct selection and colony immunoblot methods provide a sensitive and accurate strategy for identifying and enumerating marked organisms recovered from soil samples. We also developed a rapid assay for MOP cyclase that does not require cell permeabilization with toluene. This assay can be used to verify tagged organisms isolated by other methods or to screen large numbers of colonies for the tag following nonselective isolation.

Journal Article↗
PubMed1997

A T-DNA gene required for agropine biosynthesis by transformed plants is functionally and evolutionarily related to a Ti plasmid gene required for catabolism of agropine by Agrobacterium strains.

The mechanisms that ensure that Ti plasmid T-DNA genes encoding proteins involved in the biosynthesis of opines in crown gall tumors are always matched by Ti plasmid genes conferring the ability to catabolize that set of opines on the inducing Agrobacterium strains are unknown. The pathway for the biosynthesis of the opine agropine is thought to require an enzyme, mannopine cyclase, coded for by the ags gene located in the T(R) region of octopine-type Ti plasmids. Extracts prepared from agropine-type tumors contained an activity that cyclized mannopine to agropine. Tumor cells containing a T region in which ags was mutated lacked this activity and did not contain agropine. Expression of ags from the lac promoter conferred mannopine-lactonizing activity on Escherichia coli. Agrobacterium tumefaciens strains harboring an octopine-type Ti plasmid exhibit a similar activity which is not coded for by ags. Analysis of the DNA sequence of the gene encoding this activity, called agcA, showed it to be about 60% identical to T-DNA ags genes. Relatedness decreased abruptly in the 5' and 3' untranslated regions of the genes. ags is preceded by a promoter that functions only in the plant. Expression analysis showed that agcA also is preceded by its own promoter, which is active in the bacterium. Translation of agcA yielded a protein of about 45 kDa, consistent with the size predicted from the DNA sequence. Antibodies raised against the agcA product cross-reacted with the anabolic enzyme. These results indicate that the agropine system arose by a duplication of a progenitor gene, one copy of which became associated with the T-DNA and the other copy of which remained associated with the bacterium.

Agrobacterium tumefaciens↗
PubMed1997

Characterization of the acc operon from the nopaline-type Ti plasmid pTiC58, which encodes utilization of agrocinopines A and B and susceptibility to agrocin 84.

The acc locus from the Ti plasmid pTiC58 confers utilization of and chemotaxis toward agrocinopines A and B (A+B), as well as susceptibility to a highly specific antiagrobacterial antibiotic, agrocin 84. DNA sequence analyses revealed that acc is composed of eight open reading frames, accR and accA through accG. Previous work showed that accR encodes the repressor which regulates this locus, and accA codes for the periplasmic binding protein of the agrocinopine transport system (S. Beck Von Bodman, G. T. Hayman, and S. K. Farrand, Proc. Natl. Acad. Sci. USA 89:643-647, 1992; G. T. Hayman, S. Beck Von Bodman, H. Kim, P. Jiang, and S. K. Farrand, J. Bacteriol. 175:5575-5584, 1993). The predicted proteins from accA through accE, as a group, have homology to proteins that belong to the ABC-type transport system superfamily. The predicted product of accF is related to UgpQ of Escherichia coli, which is a glycerophosphoryl diester phosphodiesterase, and also to agrocinopine synthase coded for by acs located on the T-DNA. The translated product of accG is related to myoinositol 1 (or 4) monophosphatases from various eucaryotes. Analyses of insertion mutations showed that accA through accE are required for transport of both agrocin 84 and agrocinopines A+B, while accF and accG are required for utilization of the opines as the sole source of carbon. Mutations in accF or accG did not abolish transport of agrocin 84, although we observed slower removal of the antibiotic from the medium by the accF mutant compared to the wild type. However, the insertion mutation in accF abolished detectable uptake of agrocinopines A+B. A mutation in accG had no effect on transport of the opines. The accF mutant was not susceptible to agrocin 84 although it took up the antibiotic. This finding suggests that agrocin 84 is activated by AccF after being transported into the bacterial cell.

ATP-Binding Cassette Transporters↗
PubMed1997

Ti plasmid conjugation is independent of vir: reconstitution of the tra functions from pTiC58 as a binary system.

Two regions of the nopaline-type Ti plasmid pTiC58 are important for conjugal transfer of this element to recipient bacteria. These two regions were cloned into two independent replicons to produce a binary transfer system. For one region, oriT/tra, we constructed two derivatives, pFRtra and pDCtra-5. Each contains the oriT site and the two flanking, divergently transcribed tra operons that encode the DNA processing functions associated with the relaxosome. These two plasmids also carry traR, which encodes the transcriptional activator necessary for expression of transfer genes. The two plasmids differ by the amounts of traB sequence or sequence downstream of traG present in the construct. The second replicon, pPLE2, carries the traI/trb region. The traI gene confers production of the Agrobacterium tumefaciens N-acyl homoserine lactone autoinducer, while the remaining genes in the trb operon encode components of the mating bridge. Donors harboring the two plasmids mobilized the transfer of the plasmid carrying the oriT/tra region to an A. tumefaciens recipient at frequencies similar to that at which the intact Ti plasmid transferred. Plasmid pFRtra, which encodes most of traB, was mobilized at a frequency almost 10-fold higher than was pDCtra-5, which lacks most of the gene. A. tumefaciens donors also mobilized pFRtra to Escherichia coli and Pseudomonas fluorescens recipients at frequencies similar to those observed with A. tumefaciens recipients. Rhizobium meliloti harboring the binary system also transferred the oriT/tra component to these recipients. However, E. coli or P. fluorescens donors harboring the binary system did not transfer pFRtra to any of the recipients. Furthermore, while the A. tumefaciens and R. meliloti donors produced high levels of the autoinducer, the P. fluorescens and E. coli donors produced only trace amounts of this signal molecule. These results indicate that the tra system of pTiC58 is fully contained within the characterized tra and trb regions of the Ti plasmid, that conjugation does not require functions encoded by the vir system for maximal activity, and that while the Ti plasmid tra system recognizes diverse gram-negative bacteria as recipients, of the hosts tested, it functions only in members of the family Rhizobiaceae.

Agrobacterium tumefaciens↗
PubMed1996

Translocation and exudation of tumor metabolites in crown galled plants.

Crown gall tumors are induced on susceptible plants by pathogenic strains of Agrobacterium. These neoplastic plants cells produce metabolites, called opines, which provide a source of nutrients to colonizing agrobacteria. Opine production previously has been shown to influence microbial communities in the immediate vicinity of the tumor. We have obtained evidence for opine translocation to and exudation from distal uninfected regions of the plant host. Grafted plants made from an opine-producing transgenic scion with a wild-type stock, or with a wild-type scion and an opine-producing stock accumulate opines in the wild-type portions of the plant. Moreover, opines were detected in root, stem, and leaf tissues of nontransgenic plants on which stem crown galls had been induced by pathogenic strains of Agrobacterium. These plants exuded opines from their roots as a component of their root exudates. Translocation of opines from the tumor to other parts of the plant, and their exudation from roots, indicates that these biologically active compounds are available to opine-catabolizing microbes that have not induced the tumors but are present in the rhizosphere or on portions of the plant distant from the site of the gall.

Biological Transport↗
PubMed1996

Ti plasmid-encoded genes responsible for catabolism of the crown gall opine mannopine by Agrobacterium tumefaciens are homologs of the T-region genes responsible for synthesis of this opine by the plant tumor.

Agrobacterium tumefaciens NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grew well with agropine (AGR) but slowly with mannopine (MOP) as the sole carbon source. When a second plasmid encoding a dedicated transport system for MOP was introduced, these cells grew well with both AGR and MOP. Transposon insertion mutagenesis and subcloning identified a 5.7-kb region of BamHI fragment 4 that encodes functions required for the degradation of MOP. DNA sequence analysis revealed seven putative genes in this region: mocD (moc for mannityl opine catabolism) and mocE, oriented from right to left, and mocRCBAS, oriented from left to right. Significant identities exist at the nucleotide and derived amino acid sequence levels between these moc genes and the mas genes that are responsible for opine biosynthesis in crown gall tumors. MocD is a homolog of Mas2, the anabolic conjugase encoded by mas2'. MocE and MocC are related to the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis. These results indicate that the moc and mas genes evolved from a common origin. MocR and MocS are related to each other and to a putative repressor for the AGR degradation system encoded by the rhizogenic plasmid pRiA4. MocB and MocA are homologs of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively. Mutations in mocD and mocE, but not mocC, are suppressed by functions encoded by the chromosome or the 450-kb megaplasmid present in many Agrobacterium isolates. We propose that moc genes derived from genes located elsewhere in the bacterial genome and that the tumor-expressed mas genes evolved from the bacterial moc genes.

Agrobacterium tumefaciens↗
PubMed1996

A Ti plasmid-encoded enzyme required for degradation of mannopine is functionally homologous to the T-region-encoded enzyme required for synthesis of this opine in crown gall tumors.

The mocC gene encoded by the octopine/mannityl opine-type Ti plasmid pTi15955 is related at the nucleotide sequence level to mas1' encoded by the T region of this plasmid. While Mas1 is required for the synthesis of mannopine (MOP) by crown gall tumor cells, MocC is essential for the utilization of MOP by Agrobacterium spp. A cosmid clone of pTi15955, pYDH208, encodes mocC and confers the utilization of MOP on strain NT1 and on strain UIA5, a derivative of NT1 lacking the 450-kb cryptic plasmid pAtC58. NT1 or UIA5 harboring pYDH208 with an insertion mutation in mocC failed to utilize MOP as the sole carbon source. Plasmid pSa-C, which encodes only mocC, complemented this mutation in both strains. This plasmid also was sufficient to confer utilization of MOP on NT1 but not on UIA5. Computer analysis showed that MocC is related at the amino acid sequence level to members of the short-chain alcohol dehydrogenase family of oxidoreductases. Lysates prepared from Escherichia coli cells expressing mocC contained an enzymatic activity that oxidizes MOP to deoxyfructosyl glutamine (santhopine [SOP]) in the presence of NAD+. The reaction catalyzed by the MOP oxidoreductase is reversible; in the presence of NADH, the enzyme reduced SOP to MOP. The apparent Km values of the enzyme for MOP and SOP were 6.3 and 1.2 mM, respectively. Among analogs of MOP tested, only N-1-(1-deoxy-D-lyxityl)-L-glutamine and N-1-(1-deoxy-D-mannityl)-L-asparagine served as substrates for MOP oxidoreductase. These results indicate that mocC encodes an oxidoreductase that, as an oxidase, is essential for the catabolism of MOP. The reductase activity of this enzyme is precisely the reaction ascribed to its T-region-encoded homolog, Mas1, which is responsible for biosynthesis of mannopine in crown gall tumors.

Agrobacterium tumefaciens↗
PubMed1996

The tra region of the nopaline-type Ti plasmid is a chimera with elements related to the transfer systems of RSF1010, RP4, and F.

The Ti plasmids of Agrobacterium tumefaciens encode two transfer systems. One mediates the translocation of the T-DNA from the bacterium to a plant cell, while the other is responsible for the conjugal transfer of the entire Ti plasmid from one bacterium to another. The determinants responsible for conjugal transfer map to two regions, tra and trb, of the nopaline-type Ti plasmid pTiC58. By using transposon mutagenesis with Tn3HoHo1, we localized the tra determinants to an 8.5-kb region that also contains the oriT region. Fusions to lacZ formed by transposon insertions indicated that this region is expressed as two divergently transcribed units. We determined the complete nucleotide sequence of an 8,755-bp region of the Ti plasmid encompassing the transposon insertions defining tra. The region contains six identifiable genes organized as two units divergently transcribable from a 258-bp inter-genic region that contains the oriT site. One unit encodes traA, traF, and traB, while the second encodes traC, traD, and traG. Reporter insertions located downstream of both sets of genes did not affect conjugation but were expressed, suggesting that the two units encode additional genes that are not involved in transfer under the conditions tested. Proteins of the predicted sizes were expressible from traA, traC, traD, and traG. The products of several Ti plasmid tra genes are related to those of other conjugation systems. The 127-kDa protein expressed from traA contains domains related to MobA of RSF1O1O and to the helicase domain of TraI of plasmid F. The translation product of traF is related to TraF of RP4, and that of traG is related to TraG of RP4 and to VirD4 of the Ti plasmid T-DNA transfer system. Genetic analysis indicated that at least traG and traF are essential for conjugal transfer, while sequence analysis predicts that traA also encodes an essential function. traB, while not essential, is required for maximum frequency of transfer. Patterns of sequence relatedness indicate that the oriT and the predicted cognate site-specific endonuclease encoded by traA share lineage with those of the transfer systems of RSF1010 and plasmid F, while genes of the Ti plasmid encoding other essential tra functions share common ancestry with genes of the RP4 conjugation system.

Agrobacterium tumefaciens↗