Experimental induction of mouthrot in Atlantic salmon smolts using Tenacibaculum maritimum from Western Canada.

Mouthrot, or bacterial stomatitis, is a disease which mainly affects farmed Atlantic salmon, (Salmo salar, L.), smolts recently transferred into salt water in both British Columbia (BC), Canada, and Washington State, USA. It is a significant fish welfare issue which results in economic losses due to mortality and antibiotic treatments. The associated pathogen is Tenacibaculum maritimum, a bacterium which causes significant losses in many species of farmed fish worldwide. This bacterium has not been proven to be the causative agent of mouthrot in BC despite being isolated from affected Atlantic salmon. In this study, challenge experiments were performed to determine whether mouthrot could be induced with T. maritimum isolates collected from outbreaks in Western Canada and to attempt to develop a bath challenge model. A secondary objective was to use this model to test inactivated whole-cell vaccines for T. maritimum in Atlantic salmon smolts. This study shows that T. maritimum is the causative agent of mouthrot and that the bacteria can readily transfer horizontally within the population. Although the whole-cell oil-adjuvanted vaccines produced an antibody response that was partially cross-reactive with several of the T. maritimum isolates, the vaccines did not protect the fish under the study's conditions.

ApoE isoform-dependent deficits in extinction of contextual fear conditioning.

The three major human apoE isoforms (apoE2, apoE3 and apoE4) are encoded by distinct alleles (ϵ2, ϵ3 and ϵ4). Compared with ϵ3, ϵ4 is associated with increased risk to develop Alzheimer's disease (AD), cognitive impairments in Parkinson's disease (PD), and other conditions. In contrast, a recent study indicated an increased susceptibility to the recurring and re-experiencing symptom cluster of Post-Traumatic Stress Disorder (PTSD), as well as related memory impairments, in patients carrying at least one ϵ2 allele. Contextual fear conditioning and extinction are used in human and animal models to study this symptom cluster. In this study, acquisition (day 1, training), consolidation (day 2, first day of re-exposure) and extinction (days 2-5) of conditioned contextual fear in human apoE2, apoE3 and apoE4 targeted replacement and C57BL/6J wild-type (WT) mice was investigated. Male and female apoE2 showed acquisition and retrieval of conditioned fear, but failed to exhibit extinction. In contrast, WT, apoE3 and apoE4 mice showed extinction. While apoE2 mice exhibited lower freezing in response to the context on day 2 than apoE3 and apoE4 mice, this cannot explain their extinction deficit as WT mice exhibited similar freezing levels as apoE2 mice on day 2 but still exhibited extinction. Elevating freezing through extended training preserved extinction in controls, but failed to ameliorate extinction deficits in apoE2 animals. These data along with clinical data showing an association of apoE2 with susceptibility to specific symptom clusters in PTSD supports an important role for apoE isoform in the extinction of conditioned fear.

An investigation on first-week mortality in layers.

The quality of day-old chick placement and management upon arrival have a major impact on first-week mortality (FWM) and subsequent welfare in layers. The present study investigated FWM and causes of FWM in 50 flocks of layers. Post mortem results from 983 chickens showed that 50% died from infections, whereas noninfectious causes, in particular dehydration and nephropathy with visceral gout, made up the remaining causes of mortality. Escherichia coli and Enterococcus faecalis were identified as the most significant bacterial pathogens associated with FWM. Statistical analysis demonstrated a significant correlation between FWM and total mortality during rearing, and a model predicting total mortality in the rearing period based on FWM was established. A statistically significant correlation between FWM and uniformity of the flock was not demonstrated at 1-2 wk of age or at approximately 15 wk of age. Genetic characterization of E. coli and E. faecalis provided evidence for a polyclonal nature of these infections in affected flocks, indicating different sources of infection. Results obtained underline the importance of minimizing FWM to a level less than 1%.

Enterococcus faecalis of human and poultry origin share virulence genes supporting the zoonotic potential of E. faecalis.

Enterococcus faecalis is a major cause of nosocomial infections in humans and has been linked to severe extra-intestinal infections in poultry. A zoonotic potential has been suggested and the aim of the present study was to investigate similarities in virulence gene profiles of E. faecalis originating from infections in humans and poultry respectively. A total of 106 isolates of E. faecalis [26 human clinical isolates, 60 poultry clinical isolates (including two small-colony variants (SCVs) and 20 poultry cloacal isolates] were investigated for presence of seven virulence-associated genes: ace, asa1, cylA, efaA, EF0591, esp and gelE. For each gene, the PCR-amplification product was sequenced from one isolate in each group to explore intragenic variations between genes of human and poultry origin. Haemolytic and protease activities were assessed and isolates were assigned a sequence type (ST). Three of the seven genes investigated (ace, efaA and gelE) were present in all isolates. The asa1 was detected in 63/80 and 13/26 isolates of poultry and human origin respectively. For cylA, the numbers were 46/80 and 14/26 respectively. Among poultry isolates, esp and EF0591 were the least frequently observed genes (1/80 and 20/80 respectively); the prevalences among human isolates were 1/26 and 18/26 respectively. A high degree of similarity between genes in human and poultry isolates were confirmed by sequencing of amplification products. None of the cylA-positive isolates demonstrated haemolytic activity, while the phenotypic expression of gelatinase varied. The ST16 was the only ST shared by human and poultry isolates. The SCV isolates did not show a unique virulence profile or phylogeny. In conclusion, regardless of the distinct phylogenetic background of most E. faecalis isolates of human and poultry origin, we found major similarities in virulence gene profile and gene sequences in isolates from the two sources, supporting the zoonotic risk associated with this organism.

Clonality and virulence traits of Escherichia coli associated with haemorrhagic septicaemia in turkeys.

Fifty-five clinical isolates of avian pathogenic Escherichia coli (APEC) from seven outbreaks of acute haemorrhagic septicaemia in turkeys were characterized by serotyping, plasmid profiling including restriction analysis with HindIII, ribotyping with EcoRI and HindIII, multilocus sequence typing (MLST) and virulence profiling. A clonal relationship was demonstrated for each outbreak according to serotype, plasmid profiling, ribotyping, and MLST. In addition, isolates demonstrated highly similar virulence profiles, as all isolates were positive for F11 pili and possessed genes encoding aerobactin (iucD), increased serum survival (iss), temperature-sensitive haemagglutinin (tsh) and colicin V plasmid operon genes (cva/cvi). However, only 20% of the isolates produced colicin V and 42% exhibited serum resistance. All strains with O group O111 and a single O18ac strain (demonstrating non-clonal DNA profiles) were positive for enteroaggregative heat-stabile toxin (EAST1), while isolates of a single outbreak all possessed the enteroaggregative toxin gene (astA). All isolates were negative for genes encoding verocytotoxins (vtx/stx), iron-repressible protein (irp2), P-fimbria (papC), invasion plasmid antigen (ipaH), attaching and effacing gene (eae), enterohaemolysin (ehxA), and enterotoxins LT, STIa (ST(p)) and STIb (ST(h)). In conclusion, highly similar virulence profiles were demonstrated for isolates of E. coli associated with a single well-defined lesion type of colibacillosis in turkeys; acute haemorrhagic septicaemia. The isolates obtained, however, demonstrated a different phylogenetic background, underlining the importance of using well-defined strain collections for characterization of APEC pathotypes.

Multi-locus sequence typing and plasmid profile characterization of avian pathogenic Escherichia coli associated with increased mortality in free-range layer flocks.

Avian pathogenic Escherichia coli strains originating from 10 free-range layer flocks were characterized by multi-locus sequence typing and plasmid profile analysis to investigate their phylogenetic relationship and diversity, respectively. In addition to colibacillosis, all flocks tested positive for antibodies against avian metapneumovirus (aMPV) during production, and six of the flocks were concurrently affected by histomonosis. Accumulated average mortality for flocks concurrently affected by colibacillosis and histomonosis made up 17.4%, while the average mortality for E. coli-infected flocks was 16.5%. A total of eight different sequence types (STs) and 47 different plasmid profiles were demonstrated among the E. coli isolates. Within each flock between one and four different STs and between three and 13 different plasmid profiles were demonstrated. A statistical significant difference in STs and plasmid profile diversity of the population of E. coli was not demonstrated between flocks affected by histomonosis compared with histomonosis-free flocks. Only minor clonal diversity was demonstrated for each flock, and in all but one flock colibacillosis started before antibodies against aMPV were detected. All isolates, except two, carried plasmids greater than 100 kb, but only a single plasmid replicon type, IncFIB, was demonstrated, suggesting plasmids representing this type might represent a common pathogenicity factor for the different STs of E. coli. Within each flock a clonal tendency was observed, indicating that only certain clones of E. coli possess a significant pathogenic potential. These clones act as primary rather than secondary pathogens, resulting in colibacillosis without predisposing factors, including histomonosis and aMPV.

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 pathway's 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.

Multiple pathways for toluene degradation in Burkholderia sp. strain JS150.

Burkholderia (Pseudomonas) sp. strain JS150 uses multiple pathways for the metabolism of catechols that result from degradation of aromatic compounds. This suggests that the strain also uses multiple upstream pathways for the initial hydroxylation of aromatic substrates. Two distinct DNA fragments that allowed Pseudomonas aeruginosa PAO1c to grow with benzene as a sole carbon source were cloned from strain JS150. One of the recombinant plasmids containing the initial steps for the degradative pathway contained a 14-kb DNA insert and was designated pRO2016. We have previously shown that the DNA insert originated from a plasmid carried by strain JS150 and contained genes encoding a multicomponent toluene-2-monooxygenase (tbmABCDEF) as well as the cognate regulatory protein (tbmR) that controls expression of the 2-monooxygenase (G. R. Johnson and R. H. Olsen, Appl. Environ. Microbiol. 61:3336-3346, 1995). Subsequently, we have identified an additional region on this DNA fragment that encodes toluene-4-monooxygenase activity. The toluene-4-monooxygenase activity was also regulated by the tbmR gene product. A second DNA fragment that allowed P. aeruginosa to grow with benzene was obtained as a 20-kb insert on a recombinant plasmid designated pRO2015. The DNA insert contained genes encoding toluene-4-monooxygenase activity but no toluene-2-monooxygenase activity. The pRO2015 insert originated from the chromosome of strain JS150, unlike the region cloned in pRO2016. Southern blots and restriction map comparisons showed that the genes for the individual 4-monooxygenases were distinct from one another. Thus, strain JS150 has been shown to have at least three toluene/benzene monooxygenases to initiate toluene metabolism in addition to the toluene dioxygenase reported previously by others.

Cross-regulation of toluene monooxygenases by the transcriptional activators TbmR and TbuT.

The toluene-3-monooxygenase from Burkholderia pickettii PKO1 and the toluene/benzene-2-monooxygenase from Burkholderia (Pseudomonas) sp. strain JS150 are distinct enzymes which differ not only in catalytic specificity and substrate range but also in the arrangement and sequence of the genes within the operons that encode the enzymes, tbuA1UBVA2C and tbmABCDEF, respectively. In the present study, we examined the transcriptional activation of the PtbuA1 and PtbmA promoters by their cognate regulators, TbuT and TbmR. TbmR and TbuT each exhibited activation of both PtbmA and PtbuA1, with toluene, benzene, and chlorobenzene serving as strong effectors. These results strongly suggest that TbmR is an NtrC-like regulator which is functionally homologous to TbuT, and they provide evidence for the evolutionary "recruitment" of the same or a similar type of regulator for both monooxygenase pathways.

Evidence for the evolution of a single component phenol/cresol hydroxylase from a multicomponent toluene monooxygenase.

We have previously reported on the organization of a unique toluene-3-monooxygenase pathway for the degradation of alkyl-substituted petroleum hydrocarbons including characteristics of the second step in the pathway transforming phenols to catechols. In the present work we have focused on the regulation and unusual genetic organization of this metabolic step. In particular, we have sequenced the 3-kb DNA interval between the region encoding the tbuD gene product (phenol/cresol hydroxylase) and part of the toluene-3-monooxygenase operon of strain PKO1. Then, various regions of this DNA were fused to a LacZ expression system to ascertain the location of the tbuD gene promoter and the binding site for its regulator, TbuT. The 5' end for transcripts for the putative promoter of the tbuD gene was also analyzed using primer extension analysis. Collectively, these results revealed that the promoter was located 2.5-kb upstream of the region encoding the tbuD gene product whose N-terminal region had been previously determined by peptide sequencing. Remarkably, the intervening 2.5-kb region showed sequence identity to results we reported previously for a multi-subunit toluene-2-monooxygenase cloned from a different bacterium, strain JS150, for which phenols are also substrates and effectors. When the DNA sequence for the tbuD gene and its contiguous 2.5-kb upstream region were compared to the entire toluene-2-monooxygenase sequence cloned from strain JS150, a promoter proximal region encoding three reading frames showed 99% identity to subunits for the toluene-2-monooxygenase operon. Within the contiguous tbuD gene region, however, DNA sequence homology was reduced to 64% overall identity and deduced amino acid sequence homology was only 21% similar. Although regions internal to the tbuD gene showed homology to corresponding toluene-2-monooxygenase subunits, domains associated with the putative functions proposed for such subunits were deleted. We believe that these results suggest that through evolution either tbuD was derived from the 2-monooxygenase pathway by deletions and molecular rearrangements, or alternatively the tbuD gene recruited part of the 2-monooxygenase pathway and its regulatory system which is activated by benzene, alkyl-substituted benzenes and phenols.

Cascade regulation of the toluene-3-monooxygenase operon (tbuA1UBVA2C) of Burkholderia pickettii PKO1: role of the tbuA1 promoter (PtbuA1) in the expression of its cognate activator, TbuT.

Burkholderia pickettii PKO1 metabolizes toluene and benzene via a chromosomally encoded toluene-3-monooxygenase pathway. Expression of the toluene-3-monooxygenase operon (tbuA1UBVA2C) is activated by the regulator, TbuT, in the presence of toluene. We have identified the TbuT coding region downstream of the toluene-3-monooxygenase structural genes by nucleotide sequence analysis and have shown that although TbuT is similar to XylR and DmpR, two members of the NtrC family of transcriptional activators which control toluene-xylene and (methyl)phenol catabolism, respectively, it is significantly different in the domain associated with effector specificity. Using a tbuA1-lacZ fusion reporter system, we determined that TbuT is activated not only by aromatic effectors but also the chlorinated aliphatic hydrocarbon trichloroethylene. Expression of tbuT and that of the tbuA1UBVA2C operon were found to be linked by readthrough transcription of tbuT from the toluene-3-monooxygenase promoter. As a result, transcription of tbuT is low when the toluene-3-monooxygenase operon is uninduced and high when expression of tbuA1UBVA2C is induced by toluene. Thus, the toluene-3-monooxygenase promoter drives the cascade expression of both the toluene-3-monooxygenase operon and tbuT, resulting in a positive feedback circuit. Examination of the nucleotide sequence upstream of the toluene-3-monooxygenase operon for promoter-like sequences revealed a -24 TGGC, -12 TTGC sequence, characteristic of sigma54 (rpoN)-dependent promoters. Primer extension and tbuA1-lacZ fusion analyses demonstrated that this -24, -12 promoter sequence, referred to as PtbuA1, was the toluene-3-monooxygenase promoter. Upstream of PtbuA1, a DNA region with dyad symmetry exhibited homology with the XylR-binding site present upstream of the Pu promoter. Deletions within this DNA sequence resulted in complete loss of expression from PtbuA1, suggesting that this region may serve as the TbuT-binding site.

Catechol 2,3-dioxygenases functional in oxygen-limited (hypoxic) environments.

We studied the degradation of toluene for bacteria isolated from hypoxic (i.e., oxygen-limited) petroleum-contaminated aquifers and compared such strains with other toluene degraders. Three Pseudomonas isolates, P. pickettii PKO1, Pseudomonas sp. strain W31, and P. fluorescens CFS215, grew on toluene when nitrate was present as an alternate electron acceptor in hypoxic environments. We examined kinetic parameters (K(m) and Vmax) for catechol 2,3-dioxygenase (C230), a key shared enzyme of the toluene-degradative pathway for these strains, and compared these parameters with those for the analogous enzymes from archetypal toluene-degrading pseudomonads which did not show enhanced, nitrate-dependent toluene degradation. C230 purified from strains W31, PKO1, and CFS215 had a significantly greater affinity for oxygen as well as a significantly greater rate of substrate turnover than found for the analogous enzymes from the TOL plasmid (pWW0) of Pseudomonas putida PaW1, from Pseudomonas cepacia G4, or from P. putida F1. Analysis of the nucleotide and deduced amino acid sequences of C23O from strain PKO1 suggests that this extradiol dioxygenase belongs to a new cluster within the subfamily of C23Os that preferentially cleave monocyclic substrates. Moreover, deletion analysis of the nucleotide sequence upstream of the translational start of the meta-pathway operon that contains tbuE, the gene that encodes the C230 of strain PKO1, allowed identification of sequences critical for regulated expression of tbuE, including a sequence homologous to the ANR-binding site of Pseudomonas aeruginosa PAO. When present in cis, this site enhanced expression of tbuE under oxygen-limited conditions. Taken together, these results suggest the occurrence of a novel group of microorganisms capable of oxygen-requiring but nitrate-enhanced degradation of benzene, toluene, ethylbenzene, and xylenes in hypoxic environments. Strain PKO1, which exemplifies this novel group of microorganisms, compensates for a low-oxygen environment by the development of an oxygen-requiring enzyme with kinetic parameters favorable to function in hypoxic environments, as well as by elevating synthesis of such an enzyme in response to oxygen limitation.

Comparison of factors influencing trichloroethylene degradation by toluene-oxidizing bacteria.

The degradation of trichloroethylene (TCE) by toluene-oxidizing bacteria has been extensively studied, and yet the influence of environmental conditions and physiological characteristics of individual strains has received little attention. To consider these effects, the levels of TCE degradation by strains distinguishable on the basis of toluene and nitrate metabolism were compared under aerobic or hypoxic conditions in the presence and absence of nitrate and an exogenous electron donor, lactate. Under aerobic conditions with toluene-induced cells, strains expressing toluene dioxygenases (Pseudomonas putida F1, Pseudomonas sp. strain JS150, Pseudomonas fluorescens CFS215, and Pseudomonas sp. strain W31) degraded TCE at low rates, with less than 12% of the TCE removed in 18 h. In contrast, strains expressing toluene monooxygenases (Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1) degraded 36 to 67% of the TCE over the same period. Under hypoxic conditions (1.7 mg of dissolved oxygen per liter) or when lactate was added as an electron donor, the extent of TCE degradation by toluene-induced cells was generally lower. In the presence of lactate, degradation of TCE by denitrifying strain PKO1 was enhanced by nitrate under conditions in which dissimilatory nitrate reduction was observed. The results of experiments performed with strains F1, G4, PKO1, and KR1 suggested that TCE or an oxidation product induces toluene degradation and that TCE induces its own degradation in the monooxygenase strains. The role of TCE as an inducer of toluene oxygenase activity in PKO1 was confirmed by performing a promoter probe analysis, in which we found that TCE activates transcription from the PKO1 3-monooxygenase operon promoter.

Nucleotide sequence analysis of genes encoding a toluene/benzene-2-monooxygenase from Pseudomonas sp. strain JS150.

It was previously shown by others that Pseudomonas sp. strain JS150 metabolizes benzene and alkyl- and chloro-substituted benzenes by using dioxygenase-initiated pathways coupled with multiple downstream metabolic pathways to accommodate catechol metabolism. By cloning genes encoding benzene-degradative enzymes, we found that strain JS150 also carries genes for a toluene/benzene-2-monooxygenase. The gene cluster encoding a 2-monooxygenase and its cognate regulator was cloned from a plasmid carried by strain JS150. Oxygen (18O2) incorporation experiments using Pseudomonas aeruginosa strains that carried the cloned genes confirmed that toluene hydroxylation was catalyzed through an authentic monooxygenase reaction to yield ortho-cresol. Regions encoding the toluene-2-monooxygenase and regulatory gene product were localized in two regions of the cloned fragment. The nucleotide sequence of the toluene/benzene-2-monooxygenase locus was determined. Analysis of this sequence revealed six open reading frames that were then designated tbmA, tbmB, tbmC, tbmD, tbmE, and tbmF. The deduced amino acid sequences for these genes showed the presence of motifs similar to well-conserved functional domains of multicomponent oxygenases. This analysis allowed the tentative identification of two terminal oxygenase subunits (TbmB and TbmD) and an electron transport protein (TbmF) for the monooxygenase enzyme. In addition to these gene products, all the tbm polypeptides shared significant homology with protein components from other bacterial multicomponent monooxygenases. Overall, the tbm gene products shared greater similarity with polypeptides from the phenol hydroxylases of Pseudomonas putida CF600, P35X, and BH than with those from the toluene monooxygenases of Pseudomonas mendocina KR1 and Burkholderia (Pseudomonas) pickettii PKO1. The relationship found between the phenol hydroxylases and a toluene-2-monooxygenase, characterized in this study for the first time at the nucleotide sequence level, suggested that DNA probes used for surveys of environmental populations should be carefully selected to reflect DNA sequences corresponding to the metabolic pathway of interest.

Physiological attributes of microbial BTEX degradation in oxygen-limited environments.

Our work has focused on the determination of physiological traits that may facilitate in situ degradation of xenobiotic compounds by indigenous microorganisms. For this our interests center on the following questions: What are the ambient conditions in a benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated aquifer? What is the behavior of indigenous bacteria under these conditions? What are the attributes of bacterial strains that are functional under hypoxic conditions? How do these strains compare with other BTEX-degrading strains?

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.

A novel toluene-3-monooxygenase pathway cloned from Pseudomonas pickettii PKO1.

Plasmid pRO1957, which contains a 26.5-kb fragment from the chromosome of Pseudomonas pickettii PKO1, allows P. aeruginosa PAO1 to grow on toluene or benzene as a sole carbon and energy source. A subclone of pRO1957, designated pRO1966, when present in P. aeruginosa PAO1 grown in lactate-toluene medium, accumulates m-cresol in the medium, indicating that m-cresol is an intermediate of toluene catabolism. Moreover, incubation of such cells in the presence of 18O2 followed by gas chromatography-mass spectrometry analysis of m-cresol extracts showed that the oxygen in m-cresol was derived from molecular oxygen. Accordingly, this suggests that toluene-3-monooxygenation is the first step in the degradative pathway. Toluene-3-monooxygenase activity is positively regulated from a locus designated tbuT. Induction of the toluene-3-monooxygenase is mediated by either toluene, benzene, ethylbenzene, or m-cresol. Moreover, toluene-3-monooxygenase activity induced by these effectors also metabolizes benzene and ethylbenzene to phenol and 3-ethylphenol, respectively, and also after induction, o-xylene, m-xylene, and p-xylene are metabolized to 3,4-dimethylphenol, 2,4-dimethylphenol, and 2,5-dimethylphenol, respectively, although the xylene substrates are not effectors. Styrene and phenylacetylene are transformed into more polar products.

Self-mobilization and organization of the genes encoding the toluene metabolic pathway of Pseudomonas mendocina KR1.

The toluene metabolic pathway of Pseudomonas mendocina KR1 is chromosomally encoded, but the pathway could be transferred by conjugation from strain KR1 to the chromosome of P. aeruginosa or P. putida. Such transconjugants utilized toluene, p-cresol, and p-hydroxybenzaldehyde. However, transconjugants were unable to further transfer toluene genes to other recipients unless Pseudomonas sex factor R68.45 was present in trans. Although the genes encoding the upper pathway for toluene metabolism in P. mendocina KR1 are sufficiently linked to permit their coordinate mobilization, they were found to be encoded in three independently regulated units: one encoding toluene-4-monooxygenase, a second encoding p-cresol methylhydroxylase and p-hydroxybenzaldehyde dehydrogenase, and a third encoding p-hydroxybenzoate hydroxylase. The last two regulatory units were cloned from the chromosome of a P. aeruginosa transconjugant onto a plasmid designated pRO1999. Analysis of pRO1999 showed that genes encoding p-cresol methylhydroxylase and p-hydroxybenzaldehyde dehydrogenase are organized as an operon; the gene encoding p-hydroxybenzaldehyde dehydrogenase is transcribed first, and this is followed by transcription of the gene encoding p-cresol methylhydroxylase. This operon is regulated by a positively acting regulator. The P. mendocina KR1 gene encoding p-hydroxybenzoate hydroxylase was linked to, but independently regulated from, the genes encoding toluene-4-monooxygenase, p-cresol methylhydroxylase, and p-hydroxybenzaldehyde dehydrogenase.

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 for in 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, or p-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 10(3) to 10(5) 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 of Pseudomonas fluorescens, designated CFS215, which exhibited nitrate dependent degradation of aromatic hydrocarbons under hypoxic conditions.