Caulobacter crescentus cells adhere to surfaces by using an extremely strong polar adhesin called the holdfast. The polysaccharide component of the holdfast is comprised in part of oligomers of N-acetylglucosamine. The genes involved in the export of the holdfast polysaccharide and the anchoring of the holdfast to the cell were previously discovered. In this study, we identified a cluster of polysaccharide biosynthesis genes (hfsEFGH) directly adjacent to the holdfast polysaccharide export genes. Sequence analysis indicated that these genes are involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide. HfsE is predicted to be a UDP-sugar lipid-carrier transferase, the glycosyltransferase that catalyzes the first step in polysaccharide biosynthesis. HfsF is predicted to be a flippase, HfsG is a glycosyltransferase, and HfsH is similar to a polysaccharide (chitin) deacetylase. In-frame hfsG and hfsH deletion mutants resulted in severe deficiencies both in surface adhesion and in binding to the holdfast-specific lectin wheat germ agglutinin. In contrast, hfsE and hfsF mutants exhibited nearly wild-type levels of adhesion and holdfast synthesis. We identified three paralogs to hfsE, two of which are redundant to hfsE for holdfast synthesis. We also identified a redundant paralog to the hfsC gene, encoding the putative polysaccharide polymerase, and present evidence that the hfsE and hfsC paralogs, together with the hfs genes, are absolutely required for proper holdfast synthesis.

An in situ technique was adopted to investigate the time-dependent ruminal degradation of chloroplast compared with recombinant DNA of Bt176 corn using conventional and quantitative PCR assays. In parallel, the Cry1Ab protein content and fragment sizes were determined by ELISA and immunoblotting techniques. Triplicate nylon bags filled with 5 g of each substrate (whole-plant isogenic, whole-plant transgenic, ensiled isogenic, and ensiled transgenic corn) were positioned within the rumen of 5 rumen-cannulated, nonlactating cows and incubated for 2, 4, 8, 16, 24, and 48 h. To investigate the DNA degradation process, PCR assays were developed to detect fragments of the endogenous highly abundant rubisco gene (173, 896, 1,197, and 1,753 bp) and of the recombinant cry1Ab gene (211, 420, 727, and 1,423 bp). Short fragments of rubisco (<431 bp) and cry1Ab DNA (211 bp) were amplifiable in whole-plant and ensiled corn samples incubated in the rumen for 48 h, whereas the traceability of larger fragments depended on previous processing of the sample (whole-plant or ensiled corn), the length of the target sequence, and concomitantly on the length of time incubated in the rumen. Quantification of rubisco and cry1Ab gene fragments applying real-time PCR assays revealed degradation to <20% of initial 0-h values within 2 h and <0.5% after 48 h of ruminal incubation. Analysis of Cry1Ab protein in whole-plant corn using the ELISA technique revealed a decrease to 28.0% of the initial value within 2 h and to 2.6% within 48 h. The concentration of Cry1Ab protein of ensiled corn was only 10% that of whole-plant corn. Ensiled corn Cry1Ab protein decreased to 10% of initial values after 48 h of ruminal incubation. Using an immunoblotting technique, the full-size Cry1Ab protein was only detectable up to 8 h; thereafter, only fragments of approximately 17 and 34 kDa size were found. In conclusion, ruminal digestion decreased the presence of functional cry1Ab gene fragments. It is unlikely that full-size, functional Cry1Ab protein will be present after 8 h of incubation in the rumen. Therefore, results based on ELISA measurements should be interpreted carefully and verified by another detection method that discriminates between the full-size and fragmented Cry1Ab protein.

Caulobacter crescentus firmly adheres to surfaces with a structure known as the holdfast, which is located at the flagellar pole of swarmer cells and at the stalk tip in stalked cells. A three-gene cluster (hfaAB and hfaC) is involved in attachment of the holdfast to the cell. Deletion and complementation analysis of the hfaAB locus revealed two genes in a single operon; both were required for holdfast attachment to the cell. Sequence analysis of the hfaAB locus showed two open reading frames with the potential to encode proteins of 15,000 and 26,000 Da, respectively. A protein migrating with an apparent size of 21 kDa in gel electrophoresis was encoded by the hfaA region when expressed in Escherichia coli under the control of the lac promoter, but no protein synthesis could be detected from the hfaB region. Sl nuclease analysis indicated that transcription of the hfaAB locus was initiated from a region containing a sequence nearly identical to the consensus for C. crescentus cr54-dependent promoters. In addition, a sequence with some similarity toftr sequences (a consensus sequence associated with other Caulobacter cr54-dependent genes) was identified upstream of the hypothesized cr-9 promoter. At least one of the hfaAB gene products was required for maximal transcription of hfaC. The sequence of hfaB showed some similarity to that of transcriptional activators of other bacteria. The C-terminal region of the putative gene product HfaA was found to be homologous to PapG and SmfG, which are adhesin molecules of enteropathogenic E. coli and Serratia marcescens, respectively. This information suggests that the protein encoded by the hfaA locus may have a direct role in the attachment of the holdfast to the cell, whereas hfaB may be involved in the positive regulation of hfaC.Caulobacter crescentus, an aquatic bacterium, is a member of microbial biofouling communities; it adheres to surfaces with an adhesive structure known as the holdfast. The holdfast is minimally a complex polysaccharide that is only found at the cell pole: at the stalk tip of stalked cells or associated with other polar structures in swarmer cells (20,25,32,35). Little is known about how the holdfast mediates adherence or why the holdfast is expressed only in the polar regions of cells. Nevertheless it is clear that holdfast expression is coordinately regulated with that of other polar organelles, appearing at the polar region of developing swarmer cell along with the flagellum, chemotactic proteins, pili, and a bacteriophage receptor (4,15,17,23,29,31,37,42). In contrast to the other developmentally regulated organelles, the holdfast and the stalk persist in the polar region, with the holdfast mediating cell attachment to surfaces for multiple generations.Much is known about the spatial and temporal regulation of synthesis of cell structures during the C. crescentus life cycle, especially with respect to the flagellar apparatus and the chemotactic proteins found in the swarmer cells (5, 27). The regulation of these pathways is a complex hierarc...

A fermentative enrichment culture (designated DHM-1) was developed that is capable of cometabolically biotransforming high concentrations of chloroform (CF) to nontoxic end products. Two Pantoea spp. were isolated from DHM-1 that also possess this dechlorination capability. Following acclimation to increasing levels of CF, corn syrup-grown DHM-1 was able to transform over 500 mg/liter CF in the presence of vitamin B 12 (approximately 3% of CF on a molar basis) at a rate as high as 22 mg/liter/day in a mineral salts medium. CO, CO 2 , and organic acids were the predominant biodegradation products, suggesting that hydrolytic reactions predominate during CF transformation. DHM-1 was capable of growing on corn syrup in the presence of high concentrations of CF (as may be present near contaminant source zones in groundwater), which makes it a promising culture for bioaugmentation. Strains DHM-1B and DHM-1T transform CF at rates similar to that of the DHM-1 enrichment culture. The ability of these strains to grow in the presence of high concentrations of CF appears to be related to alteration of membrane fluidity or homeoviscous and homeophasic adaptation.Chloroform (CF) is a toxic organic compound that is frequently detected in groundwater. In the 2007 "CERCLA Priority List of Hazardous Substances," CF ranks eleventh overall and is the third highest among chlorinated organics after vinyl chloride and polychlorinated biphenyls (4). When present at hazardous waste sites, CF is often a focal point for evaluating the feasibility of bioremediation, since it is toxic to many obligate anaerobic prokaryotes (44). For example, 1 mg/liter of CF completely inhibited dechlorination of tetrachloroethene (PCE) by a chlororespiring anaerobic isolate (32). Inhibition of reductive dechlorination of chloroethenes by CF is a general problem for sites cocontaminated with CF, which can only be overcome by first removing the CF (6).In spite of major recent advances in bioremediation of chlorinated organic compounds, treatment of CF, especially at high concentrations (e.g., Ͼ100 mg/liter), remains challenging. Although aerobic biotransformation of CF is possible (e.g., cometabolism by a butane-grown strain) (14), CF is more difficult to cometabolize than trichloroethene (42). Biotransformation of CF by mixed or pure cultures under methanogenic (5, 21) and sulfate-reducing (20) conditions has been reported, however, only at low-mg/liter CF concentrations.Corrinoids such as vitamin B 12 (i.e., cyanocobalamin) are effective catalysts for increasing the rate of halomethane biotransformation under anaerobic conditions. Addition of vitamin B 12 also shifts the pathway away from reductive dechlorination and toward hydrolytic and substitutive reactions, forming CO, CO 2 , and organic acids as the major products (8,23,24). With low levels of B 12 added (3 to 5% molar ratios of CF), an enrichment culture grown on dichloromethane (DCM) as the sole substrate (8) and a lactate-grown sulfate-reducing enrichment culture (18) were able to biotransform up ...

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The secreted endoglucanase (CenA) from the Gram-positive bacterium Cellulomonas fimi and a deletion derivative (delta CenA) lacking the N-terminal leader peptide of native CenA were used to explore the potential of delta CenA as a reporter molecule in Caulobacter crescentus. Expression of cenA in C. crescentus yielded extracellular endoglucanase activity, suggesting that the N-terminal leader peptide of CenA could direct the enzyme to the periplasm where it subsequently leaked into the medium. In contrast, expression of delta cenA yielded only cell-associated endoglucanase activity; this suggested that the enzyme retained activity in the C. crescentus cytoplasm. Using the putative cytoplasmic and periplasmic forms of delta CenA as markers, a simple assay for periplasmic delta CenA hybrids was developed. This assay indicated that delta CenA activity was largely independent of cellular location. To facilitate the use of delta CenA as a reporter, a broad host range translational fusion vector (pEC215) incorporating delta cenA was constructed. This vector was used to investigate factors important to the expression of the gene (rsaA) encoding the paracrystalline surface protein (S-layer) of the bacterium. It was found that altering the 5' untranslated region of the rsaA mRNA reduced gene expression by 70%. One rsaA:delta cenA gene fusion resulting from these experiments that incorporated only rsaA translation initiation information was further modified to serve as a general reporter for creating transcriptional gene fusions with other promoters. Gene fusions between alkaline phosphatase (phoA) and either cenA or lacZ were used to supplement information about RsaA secretion derived from rsaA:phoA gene fusions. It was found that linkage of the N-terminal leader peptide of CenA to PhoA yielded 50-100 times more cell-associated PhoA activity in C. crescentus than linkage of the RsaA N terminus. Taken together, these experiments indicated that delta CenA was useful for tagging proteins localized to the cytoplasm, exported to the periplasm, or secreted from the cell, as well as for monitoring events in the cytoplasm such as examining factors important to the level of gene expression. Further, because delta CenA was active in all cell compartments, it could be used to estimate the efficiency of hybrid protein export-secretion from enzyme activity measurements alone. In short, delta CenA possessed many of the attributes of an "all-purpose" reporter.

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