Identification and characterization of the AgmR regulator of Pseudomonas putida: role in alcohol utilization

Two-phase partitioning bioreactors (TPPBs) comprise an aqueous phase containing all non-carbon nutrients necessary for microbial growth and a solvent phase containing high concentrations of inhibitory or toxic substrates that partition at sub-inhibitory levels to the aqueous phase in response to cellular demand. This work aimed at eliminating the growth of Pseudomonas putida ATCC 11172 on medium-chain-length (C8-C12) aliphatic alcohols, hence enabling their use as xenobiotic delivery solvents within two-phase partitioning bioreactors. Experiments resulted in the isolation of a mini-Tn5 mutant unable to utilize these alcohols. The mutation, which also eliminated growth on glycerol and ethanol, was identified to be within a homologue of the P aeruginosa agmR gene, which encodes a response regulator. Enzyme analysis of the agmR::Tn5Km mutant cell extracts revealed a 10-fold decrease in pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase activity. A knockout in a gene (exaA) encoding a PQQ-linked alcohol dehydrogenase slowed but did not eliminate growth on medium-chain-length alcohols or ethanol, suggesting metabolic redundancy within P. putida ATCC 11172. Analysis of P. putida KT2440 genome sequence data indicated the presence of two PQQ-linked alcohol dehydrogenase-encoding genes. The successful elimination of alcohol utilization in the agmR mutant indicates control by AgmR on multiple pathways and presents a useful strain for biotechnological applications requiring alcohol non-utilizing microbial catalysts.     

Alcohol dehydrogenase-I from horse liver stimulates immunoglobulin production by human hybridoma and human peripheral blood lymphocytes

Immunoglobulin production stimulating activity of alcohol dehydrogenase[EC 1.1.1.1] was assessed. Alcohol dehydrogenase-I (ADH-I) derived fromhorse liver stimulated IgM production by human-human hybridoma, HB4C5 cellsproducing human lung cancer specific monoclonal IgM. IgM production of HB4C5cells was enhanced more than 6 fold by the addition of ADH-I at 400µg/ml under serum-free condition. However, yeast derived ADHs, such asADH-II and -III were ineffective to accelerate immunoglobulin production ofthe hybridoma line. These results imply that the immunoglobulin productionstimulating effect of ADH-I is irrelevant to its enzymatic function, anddefined as a novel feature of ADH-I. This enzyme also stimulated IgM and IgGproduction by human peripheral blood lymphocytes 2.9 fold and 1.4 fold,respectively . This fact suggests that ADH-I stimulates immunoglobulinproduction not only by specific hybridoma cell line, but also bynon-specific immunoglobulin producers.     

Effects of DNA on immunoglobulin production stimulating activity of alcohol dehydrogenase

Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells and lymphocytes. The IPSF activity of ADH-I was suppressed by coexistence of short DNA whose chain length is less than 200 base pairs (bp) and fibrous DNA in a dose-dependent manner. These DNA preparations completely inhibited the IPSF activity at the concentration of 250 μg/ml and 1.0 mg/ml, respectively. DNA sample termed long DNA whose average chain length is 400–7000 bp slightly stimulated IPSF activity at 0.06 μg/ml. However, long DNA suppressed IPSF activity by half at 1.0 mg/ml. The laser confocal microscopic analysis had revealed that ADH-I was incorporated by HB4C5 cells. The uptake of ADH-I was strongly inhibited by short DNA and fibrous DNA. However, long DNA did not suppress the internalization of ADH-I into HB4C5 cells. These findings indicate that short DNA and fibrous DNA depress IPSF activity of ADH-I by inhibiting the internalization of this enzyme. According to the gel-filtration analysis using HPLC, ADH-I did not directly interact with short DNA. It is expected from these findings that short DNA influences HB4C5 cells to suppress the internalization of ADH-I. Moreover, these facts also strongly suggest that ADH-I acts as IPSF after internalization into the cell. This revised version was published online in August 2006 with corrections to the Cover Date.     

The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase

The DNA sequence of the gene for the fermentative yeast alcohol dehydrogenase has been determined. The structural gene contains no introns. The amino acid sequence of the protein as determined from the nucleotide sequence disagrees with the published alcohol dehydrogenase isozyme I (ADH-I) sequence for 5 of the 347 amino acid residues. At least one, and perhaps as many as four, of these differences is probably due to ADH-I protein heterogeneity in different yeast strains and not to sequencing errors. S1 nuclease was used to map the 5' and 3' ends of the ADH-I mRNA. There are two discrete, mature 5' ends of the mRNA, mapping 27 and 37 nucleotides upstream of the translation initiating ATG. These two equally prevalent termini are 101 and 91 nucleotides, respectively, downstream from a TATAAA sequence. Analysis of the 3' end of ADH-I mRNA disclosed two minor ends upstream of the major poly(A) addition site. These three ends map 24, 67, and 83 nucleotides, respectively, downstream from the translation-terminating TAA triplet. The sequence AA-TAAG is found 28 to 34 nucleotides upstream of each ADH-I mRNA poly(A) addition site. Sequence comparisons of these three 3' ends with those for four other yeast mRNAs yielded a 13-nucleotide consensus sequence to which TAAATAAGA is central. All of the known yeast poly(A) addition sites map at or near the A residue of a CTA site 25 to 40 nucleotides downstream from this consensus octamer.     

Alcohol and aldehyde dehydrogenase from Saccharomyces cerevisiae: specific activity and influence on the production of acetic acid, ethanol and higher alcohols in the first 48 h of fermentation of grape must

The changes in the specific activity of alcohol dehydrogenase (ADH-I and ADH-II) and aldehyde dehydrogenases [AIDH-NADP+ and AIDH-NAD(P)+] from Saccharomyces cerevisiae during the first 48 h of fermentation of grape must were investigated. The biosynthesis of ADH-I and AIDH-NADP+ took place basically during the adaptation of the yeasts to the must (first 4 h), while that of ADH-II occurred immediately after exponential growth (after 12 h). From the products produced by the yeast, only the specific rate of production of ethanol was found to be directly related to the specific activity of ADH-I.     

Alielic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker

An improved method for allele replacement in Pseudomonas aeruginosa was developed. The two main ingredients of the method are: (i) novel ColE1-type cloning vectors derived from pBR322 and pUC19; and (ii) a family of cassettes containing a portable oriT, the sacB gene from Bacillus subtilis as a counter-selectable marker, and a chloramphenicol-resistance gene allowing positive selection of both oriT and sacB. Introduction of plasmid-borne DNA into the chromosome was achieved in several steps. The DNA to be exchanged was first cloned into the new ColE1-type vectors. After insertion of the oriT and sacB sequences, these plasmid were conjugally transferred into P. aeruginosa and plasmid integrants were selected. Plating on sucrose-containing medium allowed positive selection for both plasmid excision and curing since Pseudomonas aeruginosa strains containing the sacB gene in single- or multiple copy were highly sensitive to 5% sucrose in rich medium. This procedure was successfully used to introduce an agmR mutation into P. aeruginosa wild-type strain PAO1 and should allow the exchange of any DNA segment into any non-essential regions of the P. aeruginosa chromosome.     

Effects of trehalose on pressure-induced inactivation of yeast alcohol dehydrogenase

Isozymes of yeast alcohol dehydrogenase are slowly denatured at moderate hydrostatic pressures (<3 kbar). The time courses for inactivation are biphasic and both phases of both isozymes are protected by trehalose. ADH-I is slightly more barostable than ADH-II which is opposite to their thermostabilities. Trehalose at 1M extends their half-lives about 6-fold at 2 kbar, pH 7.5 and 25 degrees C. In contrast, 1M sucrose provides only 4.4-fold protection under identical conditions, a finding consistent with the superior protein stabilization of trehalose to other denaturants.     

Heterologous expression of the alcohol dehydrogenase (adhI) gene from Geobacillus thermoglucosidasius strain M10EXG

A thermostable alcohol dehydrogenase (ADH-I) isolated from the potential thermophilic ethanologen Geobacillus thermoglucosidasius strain M10EXG has been characterised. Inverse PCR showed that the gene (adhI) was localised with 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3 hexuloisomerase (PHI) on its genome. The deduced peptide sequence of the 1020-bp M10EXG adhI, which corresponds to 340 amino acids, shows 96% and 89% similarity to ADH-hT and ADH-T from Geobacillus stearothermophilus strains LLD-R and NCA 1503, respectively. Over-expression of M10EXG ADH-I in Escherichia coli DH5alpha (pNF303) was confirmed using an ADH activity assay and SDS-PAGE analysis. The specific ADH activity in the extract from this recombinant strain was 9.7(+/-0.3) U mg(-1) protein, compared to 0.1(+/-0.01) U mg(-1) protein in the control strain. The recombinant E. coli showed enzymatic activity towards ethanol, 1-butanol, 1-pentanol, 1-heptanol, 1-hexanol, 1-octanol and 2-propanol, but not methanol. In silico analysis, including phylogenetic reconstruction and protein modeling, confirmed that the thermostable enzyme from G. thermoglucosidasius is likely to belong to the NAD-Zn-dependent family of alcohol dehydrogenases.     

Occurrence of multiple forms of alcohol dehydrogenase in Penicillium supplemented with 2,3-butanediol

The NAD-dependent oxidation of ethanol, 2,3-butanediol, and other primary and secondary alcohols, catalyzed by alcohol dehydrogenases derived from Penicillium charlesii, was investigated. Alcohol dehydrogenase, ADH-I, was purified to homogeneity in a yield of 54%. The enzyme utilizes several primary alcohols as substrates, with K_m values of the order of 10^?4m. A K_m value of 60 mm was obtained for R,R,-2,3-butanediol. The stereospecificity of the oxidation of 2-butanol was investigated, and S-(+)-2-butanol was found to be oxidized 2.4 times faster than was R-(?)-2-butanol. The reduction of 2-butanone was shown to produce S-(+)-2-butanol and R-(?)-butanol in a ratio of 7:3. ADH-I is the primary isozyme of alcohol dehydrogenase present in cultures utilizing glucose as the sole carbon source. The level of alcohol dehydrogenase activity increased 7.6-fold in mycelia from cultures grown with glucose and 2,3-butanediol (0.5%) as carbon sources compared with the activity in cultures grown on only glucose. Two additional forms of alcohol dehydrogenase, ADH-II and ADH-III, were present in the cultures supplemented with 2,3-butanediol. These forms of alcohol dehydrogenase catalyze the oxidation of ethanol and 2,3-butanediol. These data suggest that P. charlesii carries out an oxidation of 2,3-butanediol which may constitute the first reaction in the degradation of 2,3-butanediol as well as the last reaction in the mixed-acid fermentation. Alcohol dehydrogenase activities in P. charlesii may be encoded by multiple genes, one which is expressed constitutively and others whose expression is inducible by 2,3-butanediol.     

Production of L-phenylacetyl carbinol by biotransformation: product and by-product formation and activities of the key enzymes in wild-type and ADH isoenzyme mutants of Saccharomyces cerevisiae

The capacities of yeast wild-type and mutants strains known to lack specific ADH isoenzymes to produce L-phenylacetyl carbinol (PAC) and benzyl alcohol in biotransformation trials were also investigated. Pyruvate decarboxylase activity, responsible for PAC formation and ADH activity, which can participate in reduction of benzaldehyde to benzyl alcohol, was also determined in each strain. In addition, the capacity of each strain to produce ethanol was investigated. Mutant strains lacking all of the isoenzymes, ADH-I, ADH-II, and ADH-III, still exhibited some ADH activity and were capable of production of benzyl alcohol and ethanol.     

Novel plasmid vectors for gene cloning in Pseudomonas.

Novel host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. We found that a new Pseudomonas strain, Pseudomonas flavida IF-4, isolated from soil, carried two small cryptic plasmids, named pNI10 and pNI20. They were multi-copy, but not self-transmissible, and the genome size was 3.7 kb for pNI10 and 2.9 kb for pNI20. Several types of cloning vectors containing a kanamycin or streptomycin resistance (Kmr or Smr) gene were constructed from pNI10 and pNI20. These plasmid vectors were efficiently transformed into several strains of Pseudomonas at a frequency up to 4 x 10(5) transformants per 1 microgram plasmid DNA by the usual competent cell method. The vectors derived from pNI10 replicated not only in Pseudomonas but also in some other Gram-negative enteric bacteria such as Escherichia coli, Enterobacter aerogenes, and Proteus mirabilis.     

Study of the reactivity of quinohemoprotein alcohol dehydrogenase with heterocycle-pentacyanoferrate(III) complexes and the electron transfer path calculations

The reactivity of alcohol dehydrogenase IIG (ADH IIG) from Pseudomonas putida HK5 with new heterocycle-pentacyanoferrate(III) complexes and hexacyanoferrate(III) was determined at pH 7.2. The pentacyanoferrate(III) complexes contained imidazole, pyrazole, pyridine, their derivatives and 2-aminobenzothiazole as the sixth ligand. The largest reactivity of the complexes with ADH IIG was estimated for the complex containing pyridine. An apparent bimolecular constant (k _ox ) for this complex was 8.7 × 10⁵ M⁻¹s⁻¹. The lowest value of k _ox was estimated for the complex with benzotriazole (k _ox = 3.1 × 10⁴ M⁻¹s⁻¹). The investigation of the hexacyanoferrate(III) enzymatic reduction rate at different ionic strength gave a single negative charge of reduced ADH IIG. Docking calculations revealed two binding sites of the complexes in ADH-IIG structure. The first one is located at the entrance to the PQQ pocket, and the second is at the site of cytochrome domain. The calculations of electron transfer (ET) path indicated that the most effective ET takes place from heme to the complex docked at the entrance to the PQQ pocket. This shortest path is constructed of amino acids Ser607 and Cys606.     

Properties of a Pseudomonas stutzeri outer membrane channel-forming protein (NosA) required for production of copper-containing N2O reductase.

A protein (NosA) in the outer membrane of Pseudomonas stutzeri that is required for copper to be inserted into N2O reductase has been extracted and purified to homogeneity. The purified protein could form channels in black lipid bilayers. Like N2O reductase, NosA contained copper and was only made anaerobically. In contrast to N2O reductase, its synthesis was repressed by exogenous copper (but not by Mn, Co, Ni, Zn, or Fe). Also in contrast to N2O reductase, NosA homologs were not immunologically detectable in Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, or other strains of P. stutzeri.     

Degradation of dibenzothiophene by Pseudomonas putida

The microbial degradation of dibenzothiophene (DBT) and other organosulphur compounds such as thiophene-2-carboxylate (T2C) is of interest for the potential desulphurization of coal. The feasibility of degradation of DBT and T2C by Pseudomonas putida and other bacteria was analysed. Pseudomonas putida oxidized sulphur from DBT in the presence of yeast extract, but it did not when DBT was the sole source of carbon.     

The 'core' and 'accessory' regulons of Pseudomonas-specific extracytoplasmic sigma factors

Pyoverdine is a fluorescent, high-affinity peptide siderophore produced by different Pseudomonas species. The genes for pyoverdine biosynthesis depend on PvdS, an extracytoplasmic sigma factor. In this issue of Molecular Microbiology, Swingle et al. demonstrate that in the plant pathogen Pseudomonas syringae PvdS not only regulates the production of pyoverdine (core regulon), but also controls expression of other genes likely to be involved in the adaptation to the environment (accessory regulon). This accessory regulon is variable, as different sets of genes seem to be recruited according to the Pseudomonas species and its specific ecological niche.     

Modified Pseudomonas agar: new differential medium for the detection/enumeration of Pseudomonas aeruginosa in mineral water.

Pseudomonas aeruginosa has been implicated as a foodborne and waterborne pathogen and is now considered a primary infectious agent. In the present study, the survival of P. aeruginosa inoculated in mineral water was evaluated by drop counts on Pseudomonas Agar Base (PAB), PAB with CN supplement X107, PAB with cetrimide, PAB with nalidixic acid, and these media with added FeSO(4). Initial counts, before starvation, were the same in all media tested. Following this period, P. aeruginosa became sensitive to PAB with added cetrimide. The addition of FeSO(4) did not improve the recovery of stressed P. aeruginosa but gave colonies a typical dark brown colour being easily differentiated from other species that can grow at 42 degrees C. The modified Pseudomonas agar medium was also tested with several P. aeruginosa strains, other species of Pseudomonas, and other genera. Only P. aeruginosa strains (pyocyanin positive) produced the typical colonies. Our results demonstrate that Pseudomonas agar with ferrous sulphate, used for the differentiation of P. aeruginosa colonies, and nalidixic acid, used as an inhibitor of Gram-positive bacteria, might be a useful medium for the detection of injured P. aeruginosa in mineral water.     

Activation of Chi recombinational hotspots by RecBCD-like enzymes from enteric bacteria

Chi sites, 5'G-C-T-G-G-T-G-G-3', enhance homologous recombination in Escherichia coli and are activated by the RecBCD enzyme. To test the ability of Chi to be activated by analogous enzymes from other bacteria, we cloned recBCD-like genes from diverse bacteria into an E. coli recBCD deletion mutant. Clones from seven species of enteric bacteria conferred to this deletion mutant recombination proficiency, Chi hotspot activity in lambda Red- Gam- vegetative crosses, and RecBCD enzyme activities, including Chi-dependent DNA strand cleavage. Three clones from Pseudomonas aeruginosa and Ps. putida conferred recombination proficiency and ATP-dependent nuclease activity, but neither Chi hotspot activity nor Chi-dependent DNA cleavage. These results imply that Chi has been conserved as a recombination-promoting signal for RecBCD-like enzymes in enteric bacteria but not in more distantly related bacteria such as Pseudomonas spp. We discuss the possibility that other, presently unknown, nucleotide sequences serve the same function as Chi in Pseudomonas spp.     

Flagellin gene sequence variation in the genus Pseudomonas

Flagellin gene (fliC) sequences from 18 strains of Pseudomonas sensu stricto representing 8 different species, and 9 representative fliC sequences from other members of the gamma sub-division of proteobacteria, were compared. Analysis was performed on N-terminal, C-terminal and whole fliC sequences. The fliC analyses confirmed the inferred relationship between P. mendocina, P. oleovorans and P. aeruginosa based on 16S rRNA sequence comparisons. In addition, the analyses indicated that P. putida PRS2000 was closely related to P. fluorescens SBW25 and P. fluorescens NCIMB 9046T, but suggested that P. putida PaW8 and P. putida PRS2000 were more closely related to other Pseudomonas spp. than they were to each other. There were a number of inconsistencies in inferred evolutionary relationships between strains, depending on the analysis performed. In particular, whole flagellin gene comparisons often differed from those obtained using N- and C-terminal sequences. However, there were also inconsistencies between the terminal region analyses, suggesting that phylogenetic relationships inferred on the basis of fliC sequence should be treated with caution. Although the central domain of fliC is highly variable between Pseudomonas strains, there was evidence of sequence similarities between the central domains of different Pseudomonas fliC sequences. This indicates the possibility of recombination in the central domain of fliC genes within Pseudomonas species, and between these genes and those from other bacteria.