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.

Thyroid hormone resistance: the role of mutational analysis.

The finding of increased thyroxine (T4) and tri-iodothyronine (T3) levels in a patient with normal or increased thyroid-stimulating hormone is unexpected and presents a differential diagnosis between a thyroid-stimulating hormone-secreting pituitary adenoma, generalized resistance to thyroid hormone (RTH) and laboratory artefact. Without careful clinical and biochemical evaluation, errors may occur in patient diagnosis and treatment. In the case of RTH, mutation of the thyroid hormone receptor beta gene results in generalized tissue resistance to thyroid hormone. As the pituitary gland shares in this tissue resistance, euthyroidism with a normal thyroid-stimulating hormone is usually maintained by increased thyroid hormones. To date, we have identified eight pedigrees in New Zealand with mutations in the thyroid hormone receptor beta gene, including two novel mutations. Mutational analysis of the thyroid hormone receptor beta gene allows definitive diagnosis of RTH, potentially avoiding the need for protracted and expensive pituitary function testing and imaging. Mutational analysis also enables family screening and may help to avoid potential misdiagnosis and inappropriate treatment.

Insulin pump-associated adverse events are common, but not associated with glycemic control, socio-economic status, or pump/infusion set type.

AIMS: While there have been many outcome-focussed studies examining insulin pump therapy, only a few have looked at potential adverse events (AEs), with none examining the relationship between AEs and pump/infusion set type, ethnicity or socio-economic status. In addition, current data on the incidence and characteristics of pump-associated AEs are confined to one paediatric centre. We aimed to describe the incidence, characteristics and potential predictors of insulin pump-associated AEs in New Zealand adults and children with T1DM. METHODS: We approached adults and families of children with T1DM on insulin pumps in four main New Zealand centres. Participants completed a questionnaire examining pump-related issues they had experienced in the preceding 12 months. RESULTS: Response rate was 64 % with 174 of 270 eligible people participating in the study. 84 % of subjects reported one or more AEs, with an overall AE incidence of 3.42 per person/year (95 % CI 3.14, 3.73). An event serious enough to require a hospital presentation occurred in 9.8 %, all but one reporting high ketones or diabetic ketoacidosis (DKA). Set/site problems were the AE most commonly reported (by 53 % of respondents), followed by cutaneous complications (43 %) and pump malfunction (38 %). Few predictors of AEs (of any type) were found; however, a negative binomial regression model found that a longer duration of pumping (p = 0.018) and age <18 years (p = 0.043) were both associated with fewer AEs (all types combined). CONCLUSIONS: Insulin pump-associated AEs are very common. However, few variables are predictive of them with no relationships seen with glycaemic control, socio-economic status, pump manufacturer or infusion set type. Based on these findings, AEs should be anticipated in both adults and children, with anticipatory patient education and training recommended for their successful and safe use.

Attempts to detect Agrobacterium tumefaciens and bacteriophage PS8 DNA in crown gall tumors by DNA-DNA-filter hybridization.

A systematic study of the DNA-DNA-filter reaction is presented which measures its ability to detect small amounts of simple DNA (bacterial or bacteriophage) in model mixtures of DNA immobilized on filters. Saturation curves show qualitatively that significant binding occurs when there is 10% Agrobacterium tumefaciens DNA on the filter but not 1%. PS8 bacteriophage DNA is detectable at a level of 0.1%. True saturation is not attained in the bacterial DNA reaction : radioactivity bound represents only 3% of the theoretical saturation value. The bacteriophage DNA reactions attain 15-30% of the expected saturation value. When crown gall tumor DNA filters were tested for the presence of A. tumefaciens or PS8 bacteriophage DNA by saturation reactions, an apparently significant amount of binding was observed compared with usual background levels for heterologous DNA filters. However thermal dissociation profiles revealed that no well-matched duplexes were formed. Normal tobacco callus DNA filters exhibited the same type of binding of labeled DNA to a similar extent (50-100% as much as tumor DNA filters). Both types of DNA-filters bound Bacillus subtilis and bacteriophage T4 DNA as efficiently as A. tumefaciens and PS8 DNA. The high non-specific background binding of labeled DNA by filters containing DNA isolated from plant tissue culture materials is ascribed to low single strand molecular weight of the filterbound DNA. This study provides no evidence for foreign DNA in crown gall tumors, and raises objections to the interpretation of the data of earlier investigators (Quetier, F., Huguet, T. and Guille, E. (1969) Biochem, Biophys. Res, Commun. 34, 128-133 and Srivastava, B.I.S. (1970) Life Sci. 9, 889-892) who claimed to detect A. tumefaciens DNA in crown gall tumors by DNA-DNA-filter hybridization.

RP4 promotion of transfer of a large Agrobacterium plasmid which confers virulence.

Introduction of RP4 plasmid into Agrobacterium tumefaciens promotes the transfer on solid medium of large virulence-associated plasmids from virulent donor strains to a plasmidless avirulent recipient. Exconjugants were selected for the ability to utilize octopine or nopaline as the sole source of arginine, traits which are coded for by virulence-associated plasmids in the strains employed here. All exconjugants retained the arginine auxotrophy of the recipient strain, and were resistant to ampicillin and kanamycin, drugs to which RP4 confers resistance. Five exconjugant clones from one cross were shown by alkaline sucrose gradient analysis to contain both RP4 plasmid and the large virulence-associated plasmid of the donor strain. All five exconjugants exhibited virulence on carrot, sunflower and kalanchoe plants. These results indicate that virulence and the ability to degrade octopine are plasmid-borne traits in A. tumefaciens strains 15955 and A6, and extend the evidence that large plasmids in A. tumefaciens are vectors of virulence genes.

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.

Localization and characterization of the region encoding catabolism of mannopinic acid from the octopine-type Ti plasmid pTi15955.

The region of the octopine-type tumor-inducing (Ti) plasmid pTi15955 encoding catabolism of mannopinic acid was localized to an 18-kilobase (kb) segment mapping between Ti plasmid coordinates 70 and 88. A subclone containing only this region normally did not allow utilization of any other mannityl opine. However, spontaneous mutants of the plasmid were isolated that conferred catabolism of agropinic acid. While the mutants remained regulated for mannopinic acid utilization, induction by this opine resulted in increased activity associated with agropinic acid catabolism. Respirometric studies and results from growth assays on mannopinic acid analogues indicated that the genes encoded on the 18-kb subclone were regulated in a wild-type fashion. By means of these analogues, spontaneous constitutive mutants were isolated. In several cases, the mutant phenotype was associated with an insertion event mapping within a 300-base pair region of the subclone insert. Although constitutive for catabolism of mannopinic acid, these mutants remained unable to catabolize any of the other mannityl opines. Segregation studies and genetic reconstitution experiments showed that one of the subclones isolated directly in Agrobacterium was actually composed of two complementing recombinant plasmids contained in the same cell. This indicated that the genes encoding mannopinic acid catabolism were organized into at least two complementation groups. These results point to a high degree of complexity in the organization and regulation of Ti plasmid genes encoding catabolism of a relatively simple carbon source.

Construction of a derivative of Agrobacterium tumefaciens C58 that does not mutate to tetracycline resistance.

Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker. Such mutations are associated with a resistant gene unit, tetC58, that is present in the genome of this strain. By deleting the tetC58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance. The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.

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.

A second T-region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid.

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadoriopines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 co-inherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.

Intracellular accumulation of mannopine, an opine produced by crown gall tumors, transiently inhibits growth of Agrobacterium tumefaciens.

pYDH208, a cosmid clone from the octopine-mannityl opine-type tumor-inducing (Ti) plasmid pTi15955 confers utilization of mannopine (MOP) and agropine (AGR) on Agrobacterium tumefaciens strain NT1. NT1 harboring pYDH208 with an insertion mutation in mocC, which codes for MOP oxidoreductase, not only fails to utilize MOP as a sole carbon source, but also was inhibited in its growth by MOP and AGR. In contrast, the growth of mutants with insertions in other tested moc genes was not inhibited by either opine. Growth of strains NT1 or UIA5, a derivative of C58 that lacks pAtC58, was not inhibited by MOP, but growth of NT1 or UIA5 harboring pRE10, which codes for the MOP transport system, was inhibited by the opine. When a clone expressing mocC was introduced, the growth of strain NT1(pRE10) was not inhibited by MOP, although UIA5(pRE10) was still weakly inhibited. In strain NT1(pRE10, mocC), santhopine (SOP), produced by the oxidation of MOP by MocC, was further degraded by functions encoded by pAtC58. These results suggest that MOP and, to a lesser extent, SOP are inhibitory when accumulated intracellularly. The growth of NT1(pRE10), as measured by turbidity and viable cell counts, ceased upon the addition of MOP but restarted in a few hours. Regrowth was partly the result of the outgrowth of spontaneous MOP-resistant mutants and partly the adaptation of cells to MOP in the medium. Chrysopine, isochrysopine, and analogs of MOP in which the glutamine residue is substituted with other amino acids were barely taken up by NT1(pRE10) and were not inhibitory to growth of the strain. Sugar analogs of MOP were inhibitory, and those containing sugars in the D form were more inhibitory than those containing sugars in the L form. MOP analogs containing hexose sugars were more inhibitory than those containing sugars with three, four, or five carbon atoms. Mutants of NT1(pRE10) that are resistant to MOP arose in the zone of growth inhibition. Genetic and physiological analyses indicate that the mutations are located on pRE10 and abolish uptake of the opine.

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.

Waldenström's macroglobulinaemia secreting a paraprotein with lupus anticoagulant activity: possible association with gastrointestinal tract disease and malabsorption.

A 51 year old man with Waldenström's macroglobulinaemia presented with a malabsorptive syndrome related to extensive small bowel lymphangiectasia caused by immunoglobulin accumulation. The patient's plasma had strong lupus anticoagulant activity and the IgM lambda paraprotein displayed specificity for the negatively charged phospholipids phosphatidyl serine and phosphatidyl inositol, as well as the neutral phosphatidic acid. Despite treatment for the macroglobulinaemia the patient died and at necropsy was found to have myocardial ischaemia and segmental infarcts in the spleen and kidney. The coexistence of these relatively rare findings suggests a possible association between Waldenström's macroglobulinaemia with gastrointestinal manifestations and paraprotein specificity for phospholipid.

An R plasmid of broad host-range, coding for resistance to nine antimicrobial agents endemic in Gram-negative nosocomial isolates.

Of 3952 clinical isolates of Enterobacteriaceae, 246 exhibited resistance to at least carbenicillin, gentamicin and tobramycin. All these isolates, representing eight genera, were resistant to at least nine antimicrobial agents in common, including the three key antibiotics and streptomycin, kanamycin, sisomycin, ampicillin, cephalothin and sulphonamide. The strains could be subdivided into seven groups depending upon additional resistance traits and some were resistant to as many as 15 antibiotics. When mated with a standard strain of Escherichia coli, 85% of 123 randomly selected donors transferred resistance to at least the nine core antibiotics. Some donors occasionally transferred resistance to two additional antibiotics, neomycin and tetracycline, while one Citrobacter freundi donor always transferred linked resistance to all 11 drugs. Although many donors were found to harbour more than one species of plasmid DNA, all except a strain of C. freundi contained at least a plasmid of mol. wt 89 x 10(6). Analysis of E. coli transconjugants showed this plasmid to be responsible for transferable resistance to the nine core antibiotics. Restriction-endonuclease analysis indicates that the 89 x 10(6) plasmids originating from different isolates were essentially identical with each other. These results show that a particular R plasmid has established itself among the Enterobacteriaceae at Hines VA Hospital. This R plasmid appears to be the predominant genetic element responsible for linked resistance to carbenicillin, gentamicin and tobramycin among these hospital-associated bacteria.

Expression of multiple eukaryotic genes from a single promoter in Nicotiana.

We engineered an expression unit composed of three eukaryotic genes driven by a single plant-active promoter and demonstrated functional expression in planta. The individual genes were linked as translational fusions to produce a polyprotein using spacer sequences encoding specific heptapeptide cleavage recognition sites for NIa protease of tobacco vein mottling virus (TVMV). The NIa gene itself was included as the second gene of the multi-gene unit. The first and third genes, obtained from the TR region of pTi15955, encoded enzymatic functions associated with the mannityl opine biosynthetic pathway. The mannityl opine conjugase gene (mas2) was the first unit of the construct and provided the native plant-active promoter and 5' untranslated regulatory sequence. The third gene (mas1), encoding the mannityl opine reductase, furnished the native 3' untranslated region. Cis-processing of the polyprotein by the NIa protease domain was demonstrated in vitro using rabbit reticulocyte lysate and wheat germ cell-free translation systems. Tobacco plant cells transformed with the multi-gene unit produced detectable levels of mannopine, mannopinic acid, and their biosynthetic intermediates, deoxyfructosyl-glutamate and deoxyfructosyl-glutamine. This indicates that the polygene construct results in a set of functional enzymatic activities that constitute a complete metabolic pathway.

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.

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.

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

Volume 63, no. 2, p. 602: the article title should read as shown above. [This corrects the article on p. 602 in vol. 63.].