Identification and functional characterization of an ABC transport system involved in polysaccharide export of A-band lipopolysaccharide in Pseudomonas aeruginosa.

Pseudomonas aeruginosa coexpresses two distinct lipopolysaccharide (LPS) molecules known as A band and B band. B band is the serospecific LPS, while A band is the common LPS antigen composed of a D-rhamnose O-polysaccharide region. An operon containing eight genes responsible for A-band polysaccharide biosynthesis and export has recently been identified and characterized (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, unpublished data; H. L. Rocchetta, J. C. Pacan, and J. S. Lam, unpublished data). In this study, we report the characterization of two genes within the cluster, designated wzm and wzt. The Wzm and Wzt proteins have predicted sizes of 29.5 and 47.2 kDa, respectively, and are homologous to a number of proteins that comprise ABC (ATP-binding cassette) transport systems. Wzm is an integral membrane protein with six potential membrane-spanning domains, while Wzt is an ATP-binding protein containing a highly conserved ATP-binding motif. Chromosomal wzm and wzt mutants were generated by using a gene replacement strategy in P. aeruginosa PAO1 (serotype 05). Western blot analysis and immunoelectron microscopy using A-band- and B-band-specific monoclonal antibodies demonstrated that the wzm and wzt mutants were able to synthesize A-band polysaccharide, although transport of the polymer to the cell surface was inhibited. The inability of the polymer to cross the inner membrane resulted in the accumulation of cytoplasmic A-band polysaccharide. This A-band polysaccharide is likely linked to a carrier lipid molecule with a phenol-labile linkage. Chromosomal mutations in wzm and wzt were found to have no effect on B-band LPS synthesis. Rather, immunoelectron microscopy revealed that the presence of A-band LPS may influence the arrangement of B-band LPS on the cell surface. These results demonstrate that A-band and B-band O-antigen assembly processes follow two distinct pathways, with the former requiring an ABC transport system for cell surface expression.     

Targeted disruption of the TGA3 locus in Arabidopsis thaliana

A major drawback to study gene functions in plant systems is the lack of an effective gene knockout strategy. With a large number of plant genes isolated and the accelerating pace by which this collection is growing, the need for their functional analyses at the whole plant level has become increasingly urgent. Here evidence is reported for the first successful disruption of a non-selectable gene in Arabidopsis thaliana by creating a mutant of the TGA3 locus via targeted insertion of the bacterial neo gene conferring kanamycin (Km) resistance. A β-glucuronidase (GUS) expression unit outside the region of homology was used as a screenable marker to distinguish homologous recombination events from those of ectopic insertions. PCR amplification coupled with Southern blot screening identified two putative homologous recombination events among 2580 Km^r calli. One callus line was subsequently isolated and the structure of the targeted TGA3 allele confirmed by Southern blot analyses. This study demonstrates the feasibility of targeting a non-selectable locus in Arabidopsis. Combined with future improvements in negative selection strategies and efficient, transformation methodologies, gene replacement studies in plants could become a routine technique.     

Use of Adenosine 5′-Triphosphate as an Indicator of the Microbiota Biomass in Rumen Contents

A number of techniques were tested for their efficiency in extracting adenosine 5′-triphosphate (ATP) from strained rumen fluid (SRF). Extraction with 0.6 N H₂SO₄, using a modification of the procedure described by Lee et al. (1971), was the most efficient and was better suited for extracting particulate samples. Neutralized extracts could not be stored frozen before assaying for ATP because large losses were incurred. The inclusion of internal standards was necessary to correct for incomplete recovery of ATP. The ATP concentration in rumen contents from a cow receiving a ration of dried roughage (mainly alfalfa hay) ranged from 31 to 56 μg of ATP per g of contents. Approximately 75% of the ATP was associated with the particulate material. The ATP was primarily of microbial origin, since only traces of ATP were present in the feed and none was found in “cell-free” rumen fluid. Fractionation of the bacterial and protozoal populations in SRF resulted in the isolation of an enriched protozoal fraction with a 10-fold higher ATP concentration than that of the separated rumen bacteria. The ATP pool sizes of nine functionally important rumen bacteria during the exponential phase of growth ranged from 1.1 to 17.6 μg of ATP per mg of dry weight. This information indicates that using ATP as a measure of microbial biomass in rumen contents must be done with caution because of possible variations in the efficiency of extraction of ATP from rumen contents and differences in the concentration of ATP in rumen microbes.     

Author index for volume 202

A procedure is described for a simple two-step purification of human liver propionyl-CoA carboxylase. The method is based on acid and carbon tetrachloride extraction to remove other biotin carboxylases followed by an 800-fold purification through biotin-pretreated, monomeric avidin-Sepharose 4B-CL with elution of active enzyme using a biotin gradient. The enzyme had a sedimentation coefficient of 17.4 S and polyacrylamide gel electrophoresis after reduction and alkylation revealed two nonidentical polypeptide chains of 75,000 and 60,000 M_r. The heavier chain was identified as the biotin-containing subunit by electrophoresis after avidin binding.     

Structural Analysis of the dur Loci in S. CEREVISIAE: Two Domains of a Single Multifunctional Gene

In Saccharomyces cerevisiae, the degradation of urea to carbon dioxide and ammonia is catalyzed by urea carboxylase and allophanate hydrolase. The loci coding for these enzymes (dur1 and dur2) are very tightly linked on the right arm of chromosome II between pet11 and met8. Pleiotropic mutations that fail to complement mutations in either of the dur loci were found to be predominantly located in or near the dur2 locus. We interpret these data as suggesting that the two dur loci might in reality be domains of a single gene that codes for a multifunctional polypeptide. In view of this conclusion, we have renamed the dur loci as the dur1,2 locus.     

Identification of the C. coli dnaK (groPC756) gene product.

Summary The E. coli dnaK (groPC756) gene product is essential for bacteriophage DNA replication. Bacterial DNA segments carrying this gene have been cloned onto a bacteriophage vector. The product of the dnaK gene has been identified on SDS polyacrylamide gels after infection of UV-irradiated E. coli cells. The dnaK gene codes for a polypeptide with an apparent molecular weight of 93,000-Mr. Transducing phages carrying amber mutations in the dnaK gene fail to induce the synthesis of the 93,000-Mr polypeptide chain upon infection of sup ⁺ bacteria, but do so upon infection of supF bacteria. E. coli carrying the dnaK756 mutation are, in addition, temperature sensitive for growth at 43° C. It is shown that the dnaK756 mutation results in an overproduction of the dnaK gene product at that temperature.     

Chemical synthesis of [32P]pyrophosphate with high specific activity

Graphical methods have traditionally been the principal means for estimation of parameters (e.g., affinity constants, cooperativity parameters, and concentrations of receptor sites) in enzymology and ligand-binding problems. The present report provides a review of these methods as well as new results, as applied to three coordinate systems popularly used in ligand-binding studies: $\text{B}\text{F}$ vs [Bound]. $\text{B}\text{F}$ vs [Free], and $\text{B}\text{F}$ vs [Total]. We consider two extremely general models, the statistical mechanical model and the Adair model for equilibrium ligand binding. We also consider a very specialized case of receptor interaction wherein the equilibrium constannt of dissociation is linearly related to receptor occupancy. We collect previously described equations and derive new ones, to enable the user to estimate the parameters of the models in terms of relatively easily measurable graphical characteristics. We have evaluated the performance of these methods in representative cases using Monte Carlo studies. The results indicate the kind of precision and accuracy which can be obtained with typical experimental designs. Depending upon the magnitude of experimental error, the graphical methods can provide dependable values for the binding parameters. However, in general, the results obtained by the graphical methods should be regarded as reasonable initial estimates for further refinement by weighted nonlinear least-squares curve fitting.     

Photoreactions of cytochrome b6 in systems using resolved chloroplast electron-transfer complexes

Photoreactions of cytochrome b₆ have been studied using resolved chloroplast electron-transfer complexes. In the presence of Photosystem (PS) II and the cytochrome b₆-f complex, photoreduction of the cytochrome can be observed. No soluble components are required for this reaction. Cytochrome b₆ photoreduction was found to be inhibited by quinone analogs, which inhibit at the Rieske iron-sulfur center of the cytochrome complex, by the addition of ascorbate and by depletion of the Rieske center and bound plastoquinone from the cytochrome complex. Photoreduction of cytochrome b₆ can also be demonstrated in the presence of the cytochrome complex and PS I. This photoreduction requires plastocyanin and a low-potential electron donor, such as durohydroquinone. Cytochrome b₆ photoreduction in the presence of PS I is inhibited by quinone analogs which interact with the Rieske iron-sulfur center. These results are discussed in terms of a Q-cycle mechanism in which plastosemiquinone serves as the reductant for cytochrome b₆ via an oxidant-induced reductive pathway.