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L J Forney

Publications and source records attributed to L J Forney.

At least 37 records · Page 2Linked to original sources
PubMed1996

Autoradiographic method for isolation of diverse microbial species with unique catabolic traits.

A novel autoradiographic method for isolation of bacteria with unique catabolic traits was developed to overcome many of the limitations of traditional selective enrichment techniques. The method consists of five steps. (i) An environmental sample is directly plated (without enrichment) on a microporous filter atop a solid medium that allows cultivation of diverse kinds of microorganisms. (ii) Once colonies form, two replicas of the filter are prepared and the colonies are regrown. (iii) The replica filters are starved 24 to 72 h to deplete intracellular carbon reserves and then (iv) placed on Na(inf2)(sup35)SO(inf4)-containing solid media with and without a test compound. (v) Following an incubation period, the replica filters are exposed to film in order to identify colonies that incorporate more (sup35)S into cell biomass in the presence of the test compound than in its absence, providing presumptive evidence for metabolism of the compound. The colonies identified in this manner can be recovered from the master filter. To demonstrate this technique, bacteria capable of degrading benzoate were isolated from a single soil slurry by traditional enrichment as well as by autoradiography. From the enrichment culture, a single isolate able to degrade benzoate was obtained. In contrast, 18 distinct strains were obtained by purifying 19 putative benzoate-degrading colonies identified by autoradiography. Each of the 18 strains was able to completely transform the substrate, as determined by high-performance liquid chromatography analyses. The doubling times of a subset of the isolates grown in benzoate medium ranged from 1.4 to 17.1 h, whereas the doubling time of the isolate obtained by enrichment was 2.0 h. These data demonstrate that the method described here can be used to obtain a collection of diverse organisms able to metabolize a specific compound.

Journal Article↗
PubMed1996

Evidence for Acquisition in Nature of a Chromosomal 2,4-Dichlorophenoxyacetic Acid/(alpha)-Ketoglutarate Dioxygenase Gene by Different Burkholderia spp.

We characterized the gene required to initiate the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by the soil bacterium Burkholderia sp. strain TFD6, which hybridized to the tfdA gene of the canonical 2,4-D catabolic plasmid pJP4 under low-stringency conditions. Cleavage of the ether bond of 2,4-D by cell extracts of TFD6 proceeded by an (alpha)-ketoglutarate-dependent reaction, characteristic of TfdA (F. Fukumori and R. P. Hausinger, J. Bacteriol. 175:2083-2086, 1993). The TFD6 tfdA gene was identified in a recombinant plasmid which complemented a tfdA transposon mutant of TFD6 created by chromosomal insertion of Tn5. The plasmid also expressed TfdA activity in Escherichia coli DH5(alpha), as evidenced by enzyme assays with cell extracts. Sequence analysis of the tfdA gene and flanking regions from strain TFD6 showed 99.5% similarity to a tfdA gene cloned from the chromosome of a different Burkholderia species (strain RASC) isolated from a widely separated geographical area. This chromosomal gene has 77.2% sequence identity to tfdA from plasmid pJP4 (Y. Suwa, W. E. Holben, and L. J. Forney, abstr. Q-403, in Abstracts of the 94th General Meeting of the American Society for Microbiology 1994.). The tfdA homologs cloned from strains TFD6 and RASC are the first chromosomally encoded 2,4-D catabolic genes to be reported. The occurrence of highly similar tfdA genes in different bacterial species suggests that this chromosomal gene can be horizontally transferred.

Journal Article↗
PubMed1996

Characterization of a chromosomally encoded 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase from Burkholderia sp. strain RASC.

The findings of previous studies indicate that the genes required for metabolism of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) are typically encoded on broad-host-range plasmids. However, characterization of plasmid-cured strains of Burkholderia sp. strain RASC, as well as mutants obtained by transposon mutagenesis, suggested that the 2,4-D catabolic genes were located on the chromosome of this strain. Mutants of Burkholderia strain RASC unable to degrade 2,4-D (2,4-D- strains) were obtained by insertional inactivation with Tn5. One such mutant (d1) was shown to have Tn5 inserted in tfdARASC, which encodes 2,4-D/alpha-ketoglutarate dioxygenase. This is the first reported example of a chromosomally encoded tfdA. The tfdARASC gene was cloned from a library of wild-type Burkholderia strain RASC DNA and shown to express 2,4-D/alpha-ketoglutarate dioxygenase activity in Escherichia coli. The DNA sequence of the gene was determined and shown to be similar, although not identical, to those of isofunctional genes from other bacteria. Moreover, the gene product (TfdARASC) was purified and shown to be similar in molecular weight, amino-terminal sequence, and reaction mechanism to the canonical TfdA of Alcaligenes eutrophus JMP134. The data presented here indicate that tfdA genes can be found on the chromosome of some bacterial species and suggest that these catabolic genes are rather mobile and may be transferred by means other than conjugation.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1996

Capture of a catabolic plasmid that encodes only 2,4-dichlorophenoxyacetic acid:alpha-ketoglutaric acid dioxygenase (TfdA) by genetic complementation.

The modular pathway for the metabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) encoded on plasmid pJP4 of Alcaligenes eutrophus JMP134 appears to be an example in which two genes, tfdA and tfdB, have been recruited during the evolution of a catabolic pathway. The products of these genes act to convert 2,4-D to a chloro-substituted catechol that can be further metabolized by enzymes of a modified ortho-cleavage pathway encoded by tfdCDEF. Given that modified ortho-cleavage pathways are comparatively common and widely distributed among bacteria, we sought to determine if microbial populations in soil carry tfdA on plasmid vectors that lack tfdCDEF or tfdB. To capture such plasmids from soil populations, we used a recipient strain of A. eutrophus that was rifampin resistant and carried a derivative of plasmid pJP4 (called pBH501aE) in which the tfdA had been deleted. Upon mating with mixed bacterial populations from soil treated with 2,4-D, transconjugants that were resistant to rifampin yet able to grow on 2,4-D were obtained. Among the transconjugants obtained were clones that contained a ca. 75-kb plasmid, pEMT8. Bacterial hosts that carried this plasmid in addition to pBH501aE metabolized 2,4-D, whereas strains with only pEMT8 did not. Southern hybridization showed that pEMT8 encoded a gene with a low level of similarity to the tfdA gene from plasmid pJP4. Using oligonucleotide primers based on known tfdA sequences, we amplified a 330-bp fragment of the gene and determined that it was 77% similar to the tfdA gene of plasmid pJP4 and 94% similar to tfdA from Burkholderia sp. strain RASC. Plasmid pEMT8 lacked genes that exhibited significant levels of homology to tfdB and tfdCDEF. Moreover, cell extracts from A. eutrophus(pEMT8) cultures did not exhibit TfdB, TfdC, TfdD, and TfdE activities, whereas cell extracts from A. eutrophus(pEMT8)(pBH501aE) cultures did. These data suggest that pEMT8 encodes only tfdA and that this gene can effectively complement the tfdA deletion mutation of pBH501aE.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1995

Analysis of factors affecting the accuracy, reproducibility, and interpretation of microbial community carbon source utilization patterns.

We determined factors that affect responses of bacterial isolates and model bacterial communities to the 95 carbon substrates in Biolog microtiter plates. For isolates and communities of three to six bacterial strains, substrate oxidation rates were typically nonlinear and were delayed by dilution of the inoculum. When inoculum density was controlled, patterns of positive and negative responses exhibited by microbial communities to each of the carbon sources were reproducible. Rates and extents of substrate oxidation by the communities were also reproducible but were not simply the sum of those exhibited by community members when tested separately. Replicates of the same model community clustered when analyzed by principal-components analysis (PCA), and model communities with different compositions were clearly separated on the first PCA axis, which accounted for >60% of the dataset variation. PCA discrimination among different model communities depended on the extent to which specific substrates were oxidized. However, the substrates interpreted by PCA to be most significant in distinguishing the communities changed with reading time, reflecting the nonlinearity of substrate oxidation rates. Although whole-community substrate utilization profiles were reproducible signatures for a given community, the extent of oxidation of specific substrates and the numbers or activities of microorganisms using those substrates in a given community were not correlated. Replicate soil samples varied significantly in the rate and extent of oxidation of seven tested substrates, suggesting microscale heterogeneity in composition of the soil microbial community.

Journal Article↗
PubMed1995

Characterization of diverse 2,4-dichlorophenoxyacetic acid-degradative plasmids isolated from soil by complementation.

The diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative plasmids in the microbial community of an agricultural soil was examined by complementation. This technique involved mixing a suitable Alcaligenes eutrophus (Rifr) recipient strain with the indigenous microbial populations extracted from soil. After incubation of this mixture, Rifr recipient strains which grow with 2,4-D as the only C source were selected. Two A. eutrophus strains were used as recipients: JMP228 (2,4-D-), which was previously derived from A. eutrophus JMP134 by curing of the 2,4-D-degradative plasmid pJP4, and JMP228 carrying pBH501aE (a plasmid derived from pJP4 by deletion of a large part of the tfdA gene which encodes the first step in the mineralization of 2,4-D). By using agricultural soil that had been treated with 2,4-D for several years, transconjugants were obtained with both recipients. However, when untreated control soil was used, no transconjugants were isolated. The various transconjugants had plasmids with seven different EcoRI restriction patterns. The corresponding plasmids are designated pEMT1 to pEMT7. Unlike pJP4, pEMT1 appeared not to be an IncP1 plasmid, but all the others (pEMT2 to pEMT7) belong to the IncP1 group. Hybridization with individual probes for the tfdA to tfdF genes of pJP4 demonstrated that all plasmids showed high degrees of homology to the tfdA gene. Only pEMT1 showed a high degree of homology to tfdB, tfdC, tfdD, tfdE, and tfdF, while the others showed only moderate degrees of homology to tfdB and low degrees of homology to tfdC.(ABSTRACT TRUNCATED AT 250 WORDS)

2,4-Dichlorophenoxyacetic Acid↗
PubMed1995

Microbial degradation and humification of the lawn care pesticide 2,4-dichlorophenoxyacetic acid during the composting of yard trimmings.

The fate of the widely used lawn care herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) during the composting of yard trimmings consisting of primarily leaves and grass is an important unexplored question. In this study, we determined the extent of 2,4-D mineralization, incorporation into humic matter, volatilization, and sorption during the composting of yard trimmings. Yard trimmings (2:1 [wt/wt] leaves-grass) were amended with 14C-ring-labeled 2,4-D (17 mg/kg of dry weight) and composted in a temperature-controlled laboratory scale compost system. During composting, thermophilic microbes were numerically dominant, reaching a maximum of 2 x 10(11)/g. At the end of composting, 46% of the organic matter (OM) present in the yard trimmings was lost and the compost was stable, with an oxygen uptake rate of 0.09 mg of O2 per g of OM per h, and was well humified (humification index, 0.39). Mineralization of the OM temporally paralleled mineralization of 2,4-D. In the final compost, 47% of the added 2,4-D carbon was mineralized, about 23% was complexed with high-molecular-weight humic acids, and about 20% was not extractable (humin fraction). Less than 1% of the added 14C was present in water expressed from the finished compost, suggesting a low potential for leaching of 2,4-D. Very little volatilization of 2,4-D occurred during composting. It is of interest that our results indicate active mineralization of 2,4-D at composting temperatures of 60 degrees C because microbial 2,4-D degradation at thermophilic temperatures has not been previously documented.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1994

Evidence for multiple adaptive peaks from populations of bacteria evolving in a structured habitat.

Natural selection tends to promote the divergence of populations living in different environments. Even in identical environments, however, replicate populations may diverge if they find alternative adaptive solutions. We describe the evolution of 18 bacterial populations (Comamonas sp.) founded from a single progenitor genotype and propagated separately for 1000 generations in two distinct environments, one physically unstructured (mass-action liquid) and the other structured (agar surfaces). Phenotypic diversity, as reflected in colony morphology, was greater in the structured habitat than in the unstructured habitat. More importantly, the trajectories for mean fitness, as measured by competition against the common ancestor, were more divergent for populations in the structured habitat than those in the unstructured habitat. Structured environments may accelerate evolutionary diversification by promoting genetic polymorphisms within populations, thereby increasing the complexity of genetic constraints that allow divergence among replicate populations.

Adaptation, Biological↗
PubMed1994

Accuracy, reproducibility, and interpretation of Fatty Acid methyl ester profiles of model bacterial communities.

We determined the accuracy and reproducibility of whole-community fatty acid methyl ester (FAME) analysis with two model bacterial communities differing in composition by using the Microbial ID, Inc. (MIDI), system. The biomass, taxonomic structure, and expected MIDI-FAME profiles under a variety of environmental conditions were known for these model communities a priori. Not all members of each community could be detected in the composite profile because of lack of fatty acid "signatures" in some isolates or because of variations (approximately fivefold) in fatty acid yield across taxa. MIDI-FAME profiles of replicate subsamples of a given community were similar in terms of fatty acid yield per unit of community dry weight and relative proportions of specific fatty acids. Principal-components analysis (PCA) of MIDI-FAME profiles resulted in a clear separation of the two different communities and a clustering of replicates of each community from two separate experiments on the first PCA axis. The first PCA axis accounted for 57.1% of the variance in the data and was correlated with fatty acids that varied significantly between communities and reflected the underlying community taxonomic structure. On the basis of our data, community fatty acid profiles can be used to assess the relative similarities and differences of microbial communities that differ in taxonomic composition. However, detailed interpretation of community fatty acid profiles in terms of biomass or community taxonomic composition must be viewed with caution until our knowledge of the quantitative and qualitative distribution of fatty acids over a wide variety of taxa and the effects of growth conditions on fatty acid profiles is more extensive.

Journal Article↗
PubMed1994

Identification of a gene essential for piliation in Haemophilus influenzae type b with homology to the pilus assembly platform genes of gram-negative bacteria.

Haemophilus influenzae type b (Hib) pili are complex filamentous surface structures consisting predominantly of pilin protein subunits. The gene encoding the major pilin protein subunit of Hib adherence pili has been cloned and its nucleotide sequence has been determined. In order to identify specific accessory genes involved in pilus expression and assembly, we constructed isogenic Hib mutants containing insertional chromosomal mutations in the DNA flanking the pilin structural gene. These mutants were screened for pilin production, pilus expression, and hemagglutination. Pili and pilin production were assessed by immunoassays with polyclonal antisera specific for pilin and pili of Hib strain Eagan. Hemagglutination was semiquantitatively evaluated in a microtiter plate assay. Six Hib mutants produced proteins immunoreactive with antipilin antiserum but no longer produced structures reactive with antipilus antiserum. In addition, the mutants were unable to agglutinate human erythrocytes. Nucleotide sequence analysis localized the insertion sites in the six mutants to 2.5-kb open reading frame upstream of the pilin structural gene and immediately downstream of an Hib pilin chaperone gene. The amino acid sequence encoded by this open reading frame has significant homology to members of the pilus assembly platform protein family, including FhaA of Bordetella pertussis, MrkC of Klebsiella pneumoniae, and the Escherichia coli assembly platform proteins FimD and PapC. This open reading frame, designated hifC, appears to represent a gene essential to Hib pilus biogenesis that has genetic and functional similarity to the pilus platform assembly genes of other gram-negative rods.

Amino Acid Sequence↗
PubMed1993

Reactivity of antibodies against conserved regions of pilins of Haemophilus influenzae type b.

To identify epitopes on pilins of Haemophilus influenzae type b (Hib) that may also be immunologically available on assembled pili, antisera were developed against eight synthetic peptides that represent conserved and hydrophilic regions of Hib pilin. Seven of the eight peptides were immunogenic. Binding of the anti-peptide antibodies to purified pili of Hib strain Eagan was weak. However, when the purified pili were denatured by heating, binding of the anti-peptide antibodies improved considerably, suggesting that the epitopes defined by the peptides were more available for anti-peptide antibody binding on the denatured pilins than on purified pili. On Western blot analysis, strain variation was seen in the binding of some of the anti-peptide antibodies, notably those directed against peptides in the N-terminal half of the pilin. Thus, when pilins are assembled into pili, the epitopes defined by the seven immunogenic peptides appear to be altered so that binding of the anti-peptide antibodies is greatly reduced.

Amino Acid Sequence↗
PubMed1993

Paradoxical effect of pilus expression on binding of antibodies by Haemophilus influenzae.

Haemophilus influenzae type b (Hib) pili are surface proteins that are associated with the ability of Hib to attach to human epithelial cells. Like pilus expression of other bacteria, expression of Hib pili undergoes phase variation. We observed that Hib in the piliated phase (Hib p+) bound monoclonal antibodies directed against six conserved, surface-exposed, nonpilus Hib outer membrane epitopes to a greater extent than Hib in the nonpiliated phase (Hib p-). However, after extended incubation, p+ and p- cells bound these antibodies in a similar fashion. The differential in nonpilus antibody binding to p+ and p- Hib was not related to the presence of the type b capsule. In addition, Hib p+ organisms whose pilin gene was insertionally inactivated and did not produce pili and Hib in the nonpiliated phase bound the nonpilus Hib antibodies similarly. Hib p+ and p- organisms did not differ in their binding of anti-type b capsule antibody, and the binding was specific for the epitopes recognized by the antibodies. In complement-dependent bactericidal assays, the nonpilus antibodies killed Hib p+ more effectively than Hib p-. The increased binding to, and killing of, Hib p+ by a variety of nonpilus antibodies may be important for host defense against invasive Hib.

Animals↗
PubMed1992

Effect of pili-specific antibodies on the adherence of Haemophilus influenzae type b to human buccal cells.

Different strains of Haemophilus influenzae type b (Hib) produce antigenically distinct pili that mediate adherence to human buccal epithelial cells. This study determined the ability of antibodies specific for the LKP3 pili of Haemophilus influenzae type b to inhibit the adherence of Hib strain Eagan (p+). Antiserum was prepared by immunization of rabbits with pili purified from Hib strain Eagan (p+). The presence of pili-specific antibodies in the immune serum was shown by selective immunoprecipitation of pilin and by electron microscopy of cells labeled with immunogold. Immune serum, affinity-purified antibody, and Fab fragments from immune serum significantly inhibited (P less than .001) adherence of strain Eagan (p+) to human buccal epithelial cells whereas preincubation with preimmune rabbit serum or Fab fragments from preimmune serum had no significant effect on adherence. Dilution of the immune serum, affinity-purified antibodies, or Fab fragments from immune serum resulted in decreased inhibition of Hib adherence. These results indicate that antibodies specific for LKP3 pili can effectively inhibit adherence of Hib strain Eagan (p+) and probably other strains that express this pilus serotype.

Agglutination Tests↗
PubMed1991

Comparison and analysis of the nucleotide sequences of pilin genes from Haemophilus influenzae type b strains Eagan and M43.

Previous studies have demonstrated antigenic differences among the pili expressed by various strains of Haemophilus influenzae type b (Hib). In order to understand the molecular basis for these differences, the structural gene for pilin was cloned from Hib strain Eagan (p+) and the nucleotide sequence was compared to those of strains M43 (p+) and 770235 b0f+, which had been previously determined. The pilin gene of Hib strain Eagan (p+) had a 648-bp open reading frame that encoded a 20-amino-acid leader sequence followed by the 196 amino acids found in mature pilin. The translated sequence was three amino acids larger than pilins of strains M43 (p+) and 770235 b0f+ and was 78% identical and 95% homologous when conservative amino acid substitutions were considered. Differences between the amino acid sequences were not localized to any one region but rather were distributed throughout the proteins. Comparison of protein hydrophilicity profiles showed several hydrophilic regions with sequences that were conserved between strain Eagan (p+) and pilins of other Hib strains, and these regions represent potentially conserved antigenic domains. Southern blot analyses using an intragenic probe from the pilin gene of strain Eagan (p+) showed that the pilin gene was conserved among all type b and nontypeable strains of H. influenzae examined, and only a single copy was present in these strains. Homologous genes were not present in the phylogenetically related species Pasteurella multocida, Pasteurella haemolytica, and Actinobacillus pleuropneumoniae. These data indicate that the pilin gene was highly conserved among different strains of H. influenzae and that small differences in the pilin amino acid sequences account for the observed antigenic differences of assembled pili from these strains.

Amino Acid Sequence↗
PubMed1990

The complete primary structure of pilin from Haemophilus influenzae type b strain Eagan.

Adherence of Haemophilus influenzae type b (Hib) to human oropharyngeal cells is mediated by pili which are proteinaceous filaments that extend outward from the bacterial cell surface. Pili from Hib strain Eagan were purified, and the primary structure of the major subunit, pilin, was determined. Sequencing of overlapping peptides showed the mature protein to be comprised of 196 amino acids and to have an Mr of 21,152. The amino terminal sequence was found to be homologous with the sequence previously reported for Hib strain M43 and also to have significant homology to pilins of other gram-negative pathogenic bacteria. Furthermore, Hib pilin had two cysteinyl residues in the amino terminal portion of the protein which were separated by 40 residues (positions 21 and 61); a motif found in other bacterial pilins. The data show that Hib pilin has structural features common to other bacterial pilins.

Amino Acid Sequence↗