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Bacteria employ a diverse array of strategies to survive under extreme environmental conditions but maintaining these adaptations comes at an energetic cost. If energy reserves drop too low, extremophiles may enter a dormant state to persist. We estimated bacterial dormancy and identified the environmental variables influencing our activity proxy in 10 hypersaline and freshwater lakes across the Western United States. Using ribosomal RNA:DNA ratios as an indicator for bacterial activity, we found that the proportion of the community exhibiting dormancy was 16% lower in hypersaline than freshwater lakes. Based on our indicator variable multiple regression results, saltier conditions in both freshwater and hypersaline lakes increased activity, suggesting that salinity was a robust environmental filter structuring bacterial activity in lake ecosystems. To a lesser degree, higher total phosphorus concentrations reduced dormancy in all lakes. Thus, even under extreme conditions, the competition for resources exerted pressure on activity. Within the compositionally distinct and less diverse hypersaline communities, abundant taxa were disproportionately active and localized in families Microbacteriaceae (Actinobacteria), Nitriliruptoraceae (Actinobacteria), and Rhodobacteraceae (Alphaproteobacteria). Our results are consistent with the view that hypersaline communities are able to capitalize on a seemingly more extreme, yet highly selective, set of conditions and finds that extremophiles may need dormancy less often to thrive and survive.

Macrodontia is a rare dental abnormality, which can cause cosmetic concerns. Various management techniques for this condition have been documented in the literature. This case describes the initial management of macrodontia in the mixed dentition stage with the use of a minimally invasive approach to treatment. CPD/Clinical Relevance: The importance of early referral of dental abnormities is highlighted. Short- and long-term treatment options for macrodontia are described, including the impact such anomalies can have on the developing dentition.

Great Salt Lake (GSL) represents one of the world's most hypersaline environments. In this study, the archaeal and bacterial communities at the North and South arms of the lake were surveyed by cloning and sequencing the 16S rRNA gene. The sampling locations were chosen for high salt concentration and the presence of unique environmental gradients, such as petroleum seeps and high sulfur content. Molecular techniques have not been systematically applied to this extreme environment, and thus the composition and the genetic diversity of microbial communities at GSL remain mostly unknown. This study led to the identification of 58 archaeal and 42 bacterial operational taxonomic units. Our phylogenetic and statistical analyses displayed a high biodiversity of the microbial communities in this environment. In this survey, we also showed that the majority of the 16S rRNA gene sequences within the clone library were distantly related to previously described environmental halophilic archaeal and bacterial taxa and represent novel phylotypes.

The rate, progress, and limits of trichloroethylene (TCE) degradation by Ralstonia eutropha AEK301/pYK3021 whole cells were examined in the absence of aromatic induction. At TCE concentrations up to 800 &mgr;M, degradation rates were sustained until TCE was no longer detectable. The Ks and Vmax for TCE degradation by AEK301/pYK3021 whole cells were determined to be 630 &mgr;M and 22.6 nmol/min/mg of total protein, respectively. The sustained linear rates of TCE degradation by AEK301/pYK3021 up to a concentration of 800 &mgr;M TCE suggest that solvent effects are limited during the degradation of TCE and that this construct is little affected by the formation of toxic intermediates at the TCE levels and assay duration tested. TCE degradation by this strain is subject to carbon catabolite repression.

One mutant (AEK201) of Alcaligenes eutrophus JMP134 deficient in phenol metabolism was isolated by transposon mutagenesis using pSUP2021, a suicide plasmid. The 14.5 kb EcoRI fragment containing Tn5 and flanking DNA was cloned from AEK201 and used to probe a gene bank of wild type by colony hybridization. All five positive cosmids isolated rendered AEK201 to grow on phenol. The data from subcloning revealed that a trans-acting factor encoded on the 2.3 kb SalI-HindIII fragment, which is common to all cosmids, allowed the mutant to restore three enzyme activities tested (phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase). This fragment seems to act as a positive regulator on the entire phenol pathway. Another regulatory segment was subcloned from the 16.8 kb HindIII fragment on which phenol hydroxylase and catechol 2,3-dioxygenase activities were carried [Kim, Y., Ayoubi, P., and Harker, A. R. (1996) Appl. Environ. Microbiol. 62, 3227-3233]. The expression of phenol hydroxylase activity was entirely repressed in the presence of this segment in Pseudomonas aeruginosa PAO1c, but the enzyme activity was increased in A. eutrophus AEK301, suggesting that this trans-acting factor is both an activator and a repressor for phenol hydroxylase. Possible regulatory mechanisms for the phenol pathway in A. eutrophus JMP134 are discussed.

The inducing agent of the 2,4-dichlorophenoxyacetic acid (2,4-D) pathway of Alcaligenes eutrophus JMP134 (pJP4) was determined through the analysis of promoterless lacZ transcriptional fusions with tfd structural genes. (beta)-Galactosidase activity was measured in the presence and absence of 2,4-D. Fusions of the individual genes act both as reporters and disrupters of gene expression. Increases in reporter activity were expected in fusions occurring in genes which encode enzymes which function after the production of the inducing intermediate. This analysis indicates that dichloromuconate is the inducing intermediate.

Given the demonstrated phenol-dependent trichloroethylene (TCE) degradation in Alcaligenes eutrophus JMP134 (A. R. Harker and Y. Kim, Appl. Environ. Microbiol. 56:1179-1181, 1990), this work represents a purposeful effort to create a constitutive degrader of TCE. Genes responsible for phenol hydroxylase activity were identified by Tn5 transposon mutagenesis. Mutants lacked both phenol hydroxylase and catechol 2,3-dioxygenase activities. Southern blot analysis of total DNA showed that all mutants contained a single copy of Tn5 inserted in the same 11.5-kb EcoRI fragment. Complementation with a cosmid-based gene bank constructed from A. eutrophus AEK101 allowed the isolation of three recombinant cosmids carrying a common 16.8-kb HindIII fragment. Deletion and subcloning analysis localized the genes involved in phenol hydroxylase and catechol 2,3-dioxygenase activities. Partial sequence analysis of regions within the cloned phenol hydroxylase-expressing fragment shows significant homology to the oxygenase and oxidoreductase subunits of toluene-3-monooxygenase from Pseudomonas pickettii. The Tn5-induced phl mutant, carrying a recombinant plasmid expressing the phenol hydroxylase activity, degrades TCE in the absence of induction. Complete removal of TCE (50 microM) within 24 h was observed in minimal medium containing only 0.05% ethanol as a carbon source. The bacterium removed 200 microM TCE to below detectable levels within 2 days under noninducing and nonselective conditions.

Plasmid pJP4 of Alcaligenes eutrophus JMP134 encodes the degradation of 2,4-dichlorophenoxyacetic acid. A 1.2-kb BamHI-XhoI region of the restriction fragment BamHI-E has been proposed to contain the regulatory gene tfdR (A. R. Harker, R. H. Olsen, and R. J. Seidler, J. Bacteriol. 171:314-320, 1989; B. Kaphammer, J. J. Kukor, and R. H. Olsen, J. Bacteriol. 172:2280-2286, 1990). When sequenced and analyzed, the region is shown to contain two incomplete open reading frames (ORFs) positioned divergently. The complete DNA sequence for one of the two ORFs was obtained by sequencing the adjacent restriction fragment BamHI-F. The DNA sequence reveals 100% identify with the regulatory gene tfdS of pJP4. An XbaI-PstI fragment, containing the complete ORF, encodes a 32,000-Da protein which binds to the promoter regions upstream from tfdA and tfdDII. The deduced amino acid sequence of the complete ORF shows similarity with sequences of activator proteins TcbR, CatM, and CatR of the LysR family. The complete ORF represents the regulatory gene tfdR. The deduced amino acid sequence of the incomplete ORF, situated divergently from tfdR, indicates similarity to chloromuconate cycloisomerases produced by genes tfdD and tcbD of plasmids pJP4 and pP51, respectively. This ORF is identified as part of a putative isofunctional gene, tfdDII.

The bacterium Alcaligenes eutrophus JMP134(pJP4) degrades trichloroethylene (TCE) by a chromosomal phenol-dependent pathway and by the plasmid-encoded 2,4-dichlorophenoxyacetic acid pathway. The two pathways were independent and exhibited different rates of removal and capacities for quantity of TCE removed. The phenol-dependent pathway was more rapid (0.2 versus 0.06 nmol of TCE removed per min per mg of protein) and consumed all detectable TCE. The 2,4-dichlorophenoxyacetic acid-dependent pathway removed 40 to 60% of detectable TCE.

The uptake hydrogenase of chemolithotrophically grown Rhizobium japonicum was purified to apparent homogeneity with a final specific activity of 69 mumol of H2 oxidized per min per mg of protein. The procedure included Triton extraction of broken membranes and DEAE-cellulose and Sephacryl S-200 chromatographies. The purified protein contained two polypeptides separable only by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They comigrated on native polyacrylamide gels and sucrose density gradients. The molecular weights were ca. 60,000 and 30,000. Densitometric scans of the sodium dodecyl sulfate gels indicated a molar ratio of 1.03 +/- 0.03. Antiserum was developed against the 60-kilodalton polypeptide for use in hydrogenase detection by an enzyme-linked immunosorbent assay. The antiserum did not cross-react with the 30-kilodalton polypeptide. Native gel electrophoresis of Triton-extracted cells grown in the presence of 63Ni showed comigration of the hydrogenase and radioactive Ni.

Two polypeptides present in aerobic and anaerobic cultures of Escherichia coli HB101 were shown to cross-react with antibodies to the 30- and 60-kilodalton (kDa) subunits of the uptake hydrogenase of Rhizobium japonicum. The cross-reactive polypeptides in a series of different E. coli strains are of Mrs ca. 60,000 and 30,000, and both polypeptides are present in proportion to measurable hydrogen uptake (Hup) activity (r = 0.95). The 60-kDa polypeptide from E. coli HB101 comigrated on native gels with detectable Hup activity. The exact role of the 30-kDa polypeptide in E. coli is unclear. E. coli MBM7061, a natural Hup- variant, grown anaerobically or aerobically lacked detectable Hup activity and failed to cross-react with the antisera against the hydrogenase from R. japonicum. Anaerobically cultured E. coli MBM7061, however, did express formate hydrogenlyase activity, indicating that the hydrogenases involved in the oxygen-dependent activation of hydrogen and the formate-dependent evolution of hydrogen are biochemically distinct.

Eight strains of Rhizobium lacking hydrogenase uptake (Hup) activity and 17 transconjugant strains carrying the hup cosmids pHU1, pHU52, or pHU53 (G. R. Lambert, M. A. Cantrell, F. J. Hanus, S. A. Russell, K. R. Haddad, and H. J. Evans, Proc. Natl. Acad. Sci. USA, 82:3232-3236, 1985) were screened for Hup activity and the presence of immunologically detectable hydrogenase polypeptides. Crude extracts of these strains were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with affinity-purified antibodies against the two subunits of purified hydrogenase (Mr 60,000 and 30,000). Derepressed transconjugants carrying the cosmid pHU52 were Hup+ and contained detectable levels of both hydrogenase subunit polypeptides. Non-derepressed strains, Hup- parent strains, and strains carrying cosmids other than pHU52 did not express Hup activity and contained no immunologically detectable protein. These data provide further evidence for the essential involvement of the smaller (Mr 30,000) subunit in the expression of hydrogenase activity in Rhizobium japonicum and suggest that the determinants for hydrogenase subunit synthesis are present on pHU52.

The Mo-Fe protein of Azotobacter vinelandii nitrogenase was fractionated on 9.5 M urea isoelectric focusing gels and gave three distinct bands (alpha', alpha", beta'). Protein focused on nondenaturing gels gave a single brown band, which when excised and refocused on a denaturing gel gave the three-band pattern. Partial trypsin digestion of the subunits and fractionation of the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the alpha' and alpha" polypeptide moieties were the same. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha' and beta' proteins with appropriate molecular weight standards indicated Mr = 61,000 and 57,000, respectively. This is consistent with an overall alpha 2 beta 2 mass of 236,000 daltons.

The expression of cosmid-borne Bradyrhizobium japonicum hydrogenase genes in alfalfa, clover, and soybean nodules harboring Rhizobium transconjugants was studied. Cosmid pHU52 conferred hydrogen uptake (Hup) activity in both free-living bacteria and in nodules on the different plant hosts, although in nodules the instability of the cosmid resulted in low levels of Hup activity. In contrast, cosmid pHU1, which does not confer Hup activity on free-living bacteria, gave a Hup phenotype in nodules on alfalfa and soybean. Nodules formed by B. japonicum USDA 123Spc(pHU1) recycled about 90% of nitrogenase-mediated hydrogen evolution. Both subunits of hydrogenase (30- and 60-kilodalton polypeptides) were detected in enzyme-linked immunosorbent assays of bacteroid preparations from nodules harboring B. japonicum strains with pHU1 or pHU52. Neither pHU53 nor pLAFR1 conferred detectable Hup activity in either nodules or free-living bacteria. Based on the physical maps of pHU1 and pHU52, it is suggested that a 5.5-kilobase EcoRI fragment unique to pHU52 contains a gene or part of a gene required for Hup activity in free-living bacteria but not in nodules. This conclusion is supported by the observation that two Tn5 insertions in the chromosome of B. japonicum USDA 122DES obtained by marker exchange with Tn5-mutagenized pHU1 abolished Hup activity in free-living bacteria but not in nodules.

Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD). Plasmid pRO101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4. Plasmid pRO101 was transferred by conjugation to several Pseudomonas strains and to A. eutrophus AEO106, a cured isolate of JMP134. AEO106(pRO101) and some Pseudomonas transconjugants grew on TFD. Transconjugants with a chromosomally encoded phenol hydroxylase also degraded phenoxyacetic acid (PAA) in the presence of an inducer of the TFD pathway, namely, TFD or 3-chlorobenzoate. A mutant of one such phenol-degrading strain, Pseudomonas putida PPO300(pRO101), grew on PAA as the sole carbon source in the absence of inducer. This isolate carried a mutant plasmid, designated pRO103, derived from pRO101 through the deletion of a 3.9-kilobase DNA fragment. Plasmid pRO103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD. Complementation of pRO103 in trans by a DNA fragment corresponding to the fragment deleted in pRO101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pRO101. Other subcloning experiments resulted in the cloning of the tfdA monooxygenase gene on a 3.5-kilobase fragment derived from pRO101. This subclone, in the absence of other pRO101 DNA, constitutively expressed the tfdA gene and allowed PPO300 to grow on PAA. Preliminary evidence suggests that the monooxygenase activity encoded by this DNA fragment is feedback-inhibited by phenols.

To identify the structural genes for the components of Bradyrhizobium japonicum uptake hydrogenase (Mr 60,000 and 30,000), we have expressed these genes in Escherichia coli and shown that the products cross-react with antibodies to the respective hydrogenase subunits. We constructed subclones of overlapping DNA fragments from an uptake hydrogenase-complementing cosmid, pHU52 [Lambert, G. R., Cantrell, M. A., Hanus, F. J., Russell, S. A., Haddad, K. R. & Evans, H. J. (1985) Proc. Natl. Acad. Sci. USA 82, 3232-3236], in pMZ 545, a plasmid expression vector. DNA fragments inserted into one or more of the four cloning sites downstream from the E. coli lac operon promoter (Plac) on pMZ 545 generate transcriptional, but not translational, fusions. Two subclones that directed the synthesis of Mr 60,000 and 30,000 proteins in E. coli "maxicells" were identified. The DNA inserts from these subclones were then inserted down-stream of the bacteriophage lambda PL promoter on a transcriptional fusion vector. When the PL promoter was activated in vivo by heat inactivation of the temperature sensitive cI repressor of lambda in an appropriate E. coli strain, the respective fragments expressed higher levels of Mr 60,000 and 30,000 proteins that could be detected in immunoblots. These data provide direct evidence for the presence of uptake hydrogenase structural genes on the uptake hydrogenase-complementing cosmid pHU52.

The bacterioferritin from Azotobacter vinelandii exhibits properties which in ferritins from other sources are attributed to the heteropolymeric nature of the holoprotein. The native bacterioferritin displayed multiple bands on isoelectric focusing gels. On discontinuous sodium dodecyl sulfate-polyacrylamide gels, there were two subunit polypeptides of approximate Mr 21,000 and 23,000. These molecular weights were corroborated by gel filtration experiments. Peptide maps produced by partial trypsin digestion and electrophoresis showed no detectable differences between the subunits. Similarities to well-characterized mammalian ferritins and apparent anomalies in two commonly applied electrophoretic procedures are discussed.