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.

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%.

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.

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.

Conversion of mesophilic to psychrophilic bacteria.

The minimum temperature of growth of the mesophilic bacterium Pseudomonas aeruginosa has been significantly lowered from approximately 11 degrees to 0 degrees 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.

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.

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?

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.

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.

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.

Evolution of Pseudomonas R-plasmids: consequences of Tn1 insertion and resultant partial diploidy to chromosome and Tra- R-plasmid mobilization.

Tn1 transposes from pRO161, a Tra- derivative of RP1, to Pseudomonas aeruginosa sex factor FP2. The acquisition of Tn1 by FP2 results in its ability to mobilize pRO161 to other bacteria. Genetic evidence presented here suggests two sequential mechanisms. Initially, transposition of Tn1 results in trans-diploidy for the Tra+ and Tra- plasmids. This subsequently allows mobilization of the Tra- R-plasmid dependent on a host recombination mechanism. Transconjugants from this mating contain either stable cointegrate R-plasmids or aggregates resulting from dissociation of the cointegrates into a Tra+ and Tra- plasmid. These aggregates have lost at least part of Tn1 from their parent FP2:Tn1 component, but now they mobilize the tra- R-plasmid from a recombination-deficient (Rec-) genetic background as well as from Rec+ donor strains. Transconjugants from these retransfer matings are aggregates. These results suggest a contribution of transposons to R-plasmid evolution and dissemination beyond the mere acquisition of resistance to a given antibiotic.

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.

Genetic organization and regulation of a meta cleavage pathway for catechols produced from catabolism of toluene, benzene, phenol, and cresols by Pseudomonas pickettii PKO1.

Plasmid pRO1957 contains a 26.5-kb BamHI restriction endonuclease-cleaved DNA fragment cloned from the chromosome of Pseudomonas pickettii PKO1 that allows P. aeruginosa PAO1c to grow on toluene, benzene, phenol, or m-cresol as the sole carbon source. The genes encoding enzymes for meta cleavage of catechol or 3-methylcatechol, derived from catabolism of these substrates, were subcloned from pRO1957 and were shown to be organized into a single operon with the promoter proximal to tbuE. Deletion and analysis of subclones demonstrated that the order of genes in the meta cleavage operon was tbuEFGKIHJ, which encoded catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde hydrolase, 2-hydroxymuconate semialdehyde dehydrogenase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase, 4-oxalocrotonate isomerase, and 2-hydroxypent-2,4-dienoate hydratase, respectively. The regulatory gene for the tbuEFGKIHJ operon, designated tbuS, was subcloned into vector plasmid pRO2317 from pRO1957 as a 1.3-kb PstI fragment, designated pRO2345. When tbuS was not present, meta pathway enzyme expression was partially derepressed, but these activity levels could not be fully induced. However, when tbuS was present in trans with tbuEFGKIHJ, meta pathway enzymes were repressed in the absence of an effector and were fully induced when an effector was present. This behavior suggests that the gene product of tbuS acts as both a repressor and an activator. Phenol and m-cresol were inducers of meta pathway enzymatic activity. Catechol, 3-methylcatechol, 4-methylcatechol, o-cresol, and p-cresol were not inducers but could be metabolized by cells previously induced by phenol or m-cresol.

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.

Complete nucleotide sequence of tbuD, the gene encoding phenol/cresol hydroxylase from Pseudomonas pickettii PKO1, and functional analysis of the encoded enzyme.

The gene (tbuD) encoding phenol hydroxylase, the enzyme that converts cresols or phenol to the corresponding catechols, has been cloned from Pseudomonas pickettii PKO1 as a 26.5-kbp BamHI-cleaved DNA fragment, designated pRO1957, which allowed the heterogenetic recipient Pseudomonas aeruginosa PAO1c to grow on phenol as the sole source of carbon. Two subclones of pRO1957 carried in trans have shown phenol hydroxylase activity in cell extracts of P. aeruginosa. The nucleotide sequence was determined for one of these subclones, a 3.1-kbp HindIII fragment, and an open reading frame that would encode a peptide of 73 kDa was found. The size of this deduced peptide is consistent with the size of a novel peptide that had been detected in extracts of phenol-induced cells of P. aeruginosa carrying pRO1959, a partial HindIII deletion subclone of pRO1957. Phenol hydroxylase purified from phenol-plus-Casamino Acid-grown cells of P. aeruginosa carrying pRO1959 has an absorbance spectrum characteristic of a simple flavoprotein; moreover, the enzyme exhibits a broad substrate range, accommodating phenol and the three isomers of cresol equally well. Sequence comparisons revealed little overall homology with other flavoprotein hydroxylases, supporting the novelty of this enzyme, although three conserved domains were apparent.

Catabolism of aromatic biogenic amines by Pseudomonas aeruginosa PAO1 via meta cleavage of homoprotocatechuic acid.

Pseudomonas aeruginosa PAO1 catabolized the aromatic amines tyramine and octopamine through 4-hydroxyphenylacetic acid and 3,4-dihydroxyphenylacetic acid (HPA). meta ring cleavage was mediated by 3,4-dihydroxyphenylacetate 2,3-dioxygenase (HPADO), producing 2-hydroxy-5-carboxymethylmuconic semialdehyde (MSA). An NAD-dependent dehydrogenase caused the disappearance of the yellow MSA product, probably forming 2-hydroxy-5-carboxymethylmuconic acid. Induction studies with extracts from mutant cells indicated that the inducer of HPADO was HPA and/or MSA. Strains PAO1.221 (tynC1) and PAO1.303 (tynD1) have chromosomal mutations causing a deficiency in the activity necessary for conversion of 4-hydroxyphenylacetic acid to HPA. Genetic analyses showed that the mutations were in different loci. Strains PAO1.197 (tynE1) and PAO1.185 (tynF1) are deficient in HPADO and the NAD-dependent dehydrogenase, respectively. Plasmid pRO1853 was constructed by cloning approximately 7.3 kilobases of PAO1 chromosomal DNA into the BamHI site of the vector plasmid pRO1614. This recombinant plasmid complemented the tynD1, tynE1, and tynF1 mutations. A putative repressor-binding site involved in the regulation of HPADO synthesis was observed for a subcloned fragment of pRO1853. This recombinant plasmid, pRO1863, failed to complement tynE1 or tynF1 but still complemented tynD1. Another construct, pRO1887, contained 9.2 kilobases of PAO1 chromosomal DNA inserted in the PstI site of the vector pRO1727. Plasmid pRO1887 complemented only the tynC1 mutation. Mapping experiments performed with the chromosome-mobilizing plasmid R68.45 located the mutations described above in a cluster at about 35 to 40 min of the PAO1 chromosome map. The mutations were linked to the proA, thr-48, lys-9015, argF10, and argG markers.

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.

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.

Purification and properties of mutant and wild-type diaphorases from Diplococcus pneumoniae.

Optochin-resistant mutant and wild-type diaphorases were purified approximately 300-fold by a combination of batch adsorption and column chromatography with diethylaminoethyl cellulose, and were characterized with regard to their pH optima, sensitivity to optochin inhibition and heat inactivation, Michaelis constants with flavine mononucleotide (FMN) and reduced nicotinamide adenine dinucleotide (NADH), and inhibition constants with optochin hydrochloride. The pH optima of the purified diaphorases were similar, but the purified diaphorases from the optochin-resistant strains were approximately four to five times more resistant to heat inactivation at 45 C than was the wild-type diaphorase. Purified diaphorase preparations from the optochin-resistant pneumococci had greater activities per milligram of protein and were more resistant to optochin inhibition than the preparation from the optochin-sensitive pneumococcus. Michaelis constants for FMN and NADH were similar; however, the inhibition constants of the optochin-resistant diaphorases were four to eight times greater than that of the optochin-sensitive diaphorase. Optochin hydrochloride produced a noncompetitive type of inhibition with FMN as substrate but a competitive type of inhibition with NADH as substrate. Optochin hydrochloride produced an approximately 10-fold increase in the Michaelis constant for NADH. The concentration of drug required to produce this effect was, however, greater with the mutant diaphorases than with the wild-type diaphorase. Optochin hydrochloride quenched the fluorescence of riboflavine. This phenomenon did not appear to be related to the diaphorase-inhibitory activity of the drug, however, since the pH requirements of the two reactions were different. Quenching of riboflavine fluorescence by optochin hydrochloride increased with a rise in pH, whereas inhibition of diaphorase activity by optochin hydrochloride was greater at pH 6.8 than at pH 7.6.