Ronald H. Olsen

Sequence analysis of the gene cluster encoding toluene-3-monooxygenase from Pseudomonas pickettii PKO1

The nucleotide (nt) sequence and gene organization of the locus encoding the initial step of the toluene-3-monooxygenase (Tbu) pathway from Pseudomonas pickettii PKO1 has been determined. This is the first reported nt sequence for a toluene monooxygenase which hydroxylates the C-3 position of toluene. Six tightly assembled structural genes encoding several Tbu were identified and were designated tbuA1, tbuU, tbuB, tbuV, tbuA2 and tbuC. Comparison of the deduced amino acid (aa) sequences of each open reading frame (ORF) with translated sequences from the GenBank database revealed significant overall homology to peptides from the toluene-4-monooxygenase (Tmo) from Pseudomonas mendocina KR1, the multicomponent phenol hydroxylase (Dmp) from Pseudomonas sp. strain CF600 and the methane monooxygenases (Mmo) from both Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. Similarities in both size and aa sequence between the peptides from these multicomponent oxygenases and the putative peptides from Tbu suggested roles for the TbuA1, TbuB, TbuV, TbuA2 and TbuC proteins.

Characterization and Role of tbuX in Utilization of Toluene by Ralstonia pickettii PKO1

The tbu regulon of Ralstonia pickettii PKO1 encodes enzymes involved in the catabolism of toluene, benzene, and related alkylaromatic hydrocarbons. The first operon in this regulon contains genes that encode the tbu pathways initial catabolic enzyme, toluene-3-monooxygenase, as well as TbuT, the NtrC-like transcriptional activator for the entire regulon. It has been previously shown that the organization of tbuT, which is located immediately downstream of tbuA1UBVA2C, and the associated promoter (PtbuA1) is unique in that it results in a cascade type of up-regulation of tbuT in response to a variety of effector compounds. In our efforts to further characterize this unusual mode of gene regulation, we discovered another open reading frame, encoded on the strand opposite that of tbuT, 63 bp downstream of the tbuT stop codon. The 1,374-bp open reading frame, encoding a 458-amino-acid peptide, was designated tbuX. The predicted amino acid sequence of TbuX exhibited significant similarity to several putative outer membrane proteins from aromatic hydrocarbon-degrading bacteria, as well as to FadL, an outer membrane protein needed for uptake of long-chain fatty acids in Escherichia coli. Based on sequence analysis, transcriptional and expression studies, and deletion analysis, TbuX seems to play an important role in the catabolism of toluene in R. pickettii PKO1. In addition, the expression of tbuX appears to be regulated in a manner such that low levels of TbuX are always present within the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene-3-monooxygenase. This expression pattern may relate to the possible role of TbuX as a facilitator of toluene entry into the cell.

Metabolic diversity of aromatic hydrocarbon-degrading bacteria from a petroleum-contaminated aquifer

We characterized bacteria from contaminated aquifers for their ability to utilize aromatic hydrocarbons under hypoxic (oxygen-limiting) conditions (initial dissolved oxygen concentration about 2 mg/l) with nitrate as an alternate electron acceptor. This is relevant to current intense efforts to establish favorable conditions forin situ bioremediation. Using samples of granular activated carbon slurries from an operating groundwater treatment system, we isolated bacteria that are able to use benzene, toluene, ethylbenzene, orp-xylene as their sole source of carbon under aerobic or hypoxic-denitrifying conditions. Direct isolation on solid medium incubated aerobically or hypoxically with the substrate supplied as vapor yielded 103 to 105 bacteria ml−1 of slurry supernatant, with numbers varying little with respect to isolation substrate or conditions. More than sixty bacterial isolates that varied in colony morphology were purified and characterized according to substrate utilization profiles and growth condition (i.e., aerobic vs. hypoxic) specificity. Strains with distinct characteristics were obtained using benzene compared with those isolated on toluene or ethylbenzene. In general, isolates obtained from direct selection on benzene minimal medium grew well under aerobic conditions but poorly under hypoxic conditions, whereas many ethylbenzene isolates grew well under both incubation conditions. We conclude that the conditions of isolation, rather than the substrate used, will influence the apparent characteristic substrate utilization range of the isolates obtained. Also, using an enrichment culture technique, we isolated a strain ofPseudomonas fluorescens, designated CFS215, which exhibited nitrate dependent degradation of aromatic hydrocarbons under hypoxic conditions.

Conversion of Mesophilic to Psychrophilic Bacteria

The minimum temperature of growth of the mesophilic bacterium Pseudomonas aeruginosa has been significantly lowered from approximately 11� to 0�C. This shift in the minimum temperature of growth was accompanied by a corresponding decrease in the maximum temperature of growth. Transfer of this genetic characteristic by a transducing phage grown on a psychrophile or by ultraviolet mutagenesis was used to accomplish these shifts in range of growth temperature.

Identification of cDNA clones for ligninase from Phanerochaete chrysosporium using synthetic oligonucleotide probes.

Four cDNA clones for ligninase were isolated from the cDNA library (constructed into the PstI site of E. coli vector pUC9) representing 6 day-old lignin degrading culture of Phanerochaete chrysosporium by the use of three synthetic oligonucleotide probes corresponding to partial amino acid sequences of tryptic peptides of the ligninase. Each of the three probes, 14.1, 14.2 and 25, represents a mixture of 32 12- or 14-base long oligonucleotides. Three cDNA clones hybridized with probe 14.1 but not with probe 25 or 14.2, but one cDNA clone hybridized with all of the three probes. Differential hybridization studies showed that these clones are unique to 6-day poly(A) RNA, but not to 2-day poly(A) RNA.

Escherichia coli gene transfer to unrelated bacteria by a histidine operon — RP1 drug resistance plasmid complex

Summary Genetic and physical evidence indicates that the Pseudomonas aeruginosa drug resistance factor, RP1, may promote the transfer of Escherichia coli histidine genes. The RP1 mobilized histidine genes are functionally expressed in diverse Salmonella typhimurium and P . aeruginosa histidine auxotrophs. The histidine operon — RP1 complex may be transferred back to E. coli strains from Pseudomonas .

Enumeration and characterization of BTEX-degrading bacteria from hypoxic environments functional with mixed electron acceptors☆

Our work developed oat of an interest in the behaviour of bacteria resident in benzene-, toluene-, ethylbenzeneand xylene(s)-(Le., BTEX)-contamihated aquifers. We observed that such environments are oxygen-limited, yet continueus monitoring over extended periods suggested that BTEX contaminants were disappearing and the contaminant plume may be diminished. Such observations suggested that significant microbial populations were active in situ. Moreover, laboratory studies with microcosms suggested the ascendancy of bacterial populations under hypoxic (i.e., oxygen-limited) conditions whose growth and BTEX-degrading activities were associated with the reduction of nitrate , nder conditions whereby oxygen concentrations were less t ban twenty percent of saturation.

Plasmid Content of Some Oral Microorganisms Isolated from Subgingival Plaque

Eighty-five strains of bacterial species selected from the predominant cultivable dental plaque flora of patients with different periodontal pathologies were examined for their plasmid content. Microorganisms studied included: Actinomyces viscosus, A. odontolyticus, Bacteroides asaccharolyticus (B. gingivalis), B. melaninogenicus subspecies intermedius, and subspecies melaninogenicus, Capnocytophaga ochracea (B. ochraceus), and Fusobacterium nucleatum. Three B. melaninogenicus isolates showed plasmids of approximately 2.7-2.9 Mdalton (mega-dalton) molecular size. Restriction, enzyme digests of the plasmids demonstrated dissimilar patterns when electrophoresed on agarose gels. In other microorganisms, including the Actinomyces species, plasmids were not observed.