Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in soil inoculated with Pseudomonas cepacia DBO1(pRO101), Alcaligenes eutrophus AEO106(pRO101) and Alcaligenes eutrophus JMP134(pJP4): effects of inoculation level and substrate concentration

Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) by two Alcaligenes eutrophus strains and one Pseudomonas cepacia strain containing the 2,4-D degrading plasmids pJP4 or pRO101 (=pJP4::Tn1721) was tested in 50 g (wet wt) samples of non-sterile soil. Mineralization was measured as ¹⁴C-CO₂evolved during degradation of uniformly-ring-labelled ¹⁴C-2,4-D. When the strains were inoculated to a level of approximately 10⁸ CFU/g soil, between 20 and 45% of the added 2,4-D (0.05 ppm, 10 ppm or 500 ppm) was mineralized within 72 h. Mineralization of 0.05 ppm and 10 ppm, 2,4-D by the two A. eutrophus strains was identical and rapid whereas mineralization by P. cepacia DBO1(pRO101) occurred more slowly. In contrast, mineralization of 500 ppm 2,4-D by the two A. eutrophus strains was very slow whereas mineralization by P. cepacia DBO1 was more rapid. Comparison of 2,4-D mineralization at different levels of inoculation with P. cepacia DBO1(pRO101) (6×10⁴, 6×10⁶ and 1×10⁸ CFU/g soil) revealed that the maximum mineralization rate was reached earlier with the high inoculation levels than with the low level. The kinetics of mineralization were evaluated by nonlinear regression analysis using five different models. The linear or the logarithmic form of a three-half-order model were found to be the most appropriate models for describing 2,4-D mineralization in soil. In the cases in which the logarithmic form of the three-half-order model was the most appropriate model we found, in accordance with the assumptions of the model, a significant growth of the inoculated strains.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - CFU colony forming units - PTYG peptone, tryptone, yeast & glucose - DPM disintegrations per minute     

Transcription dynamics of the functional tfdA gene during MCPA herbicide degradation by Cupriavidus necator AEO106 (pRO101) in agricultural soil

A modified protocol for simultaneous extraction of RNA and DNA, followed by real-time polymerase chain reaction quantification, was used to investigate tfdA gene expression during in situ degradation of the herbicide MCPA (4-chloro-2-methylphenoxy-acetic acid) in soil. tfdA encodes an alpha-ketoglutarate-dependent dioxygenase catalysing the first step in the degradation pathway of MCPA and 2,4-D (2,4-dichlorophenoxy-acetic acid). A linear recovery of tfdA mRNA over three orders of magnitude was shown, and the tfdA mRNA level was normalized using the tfdA mRNA/DNA ratio. The density of active cells required for tfdA mRNA detection was 10(5) cells g(-1) soil. Natural soil microcosms inoculated with Cupriavidus necator (formerly Ralstonia eutropha) AEO106 (pRO101) cells were amended with four different MCPA concentrations (2, 20, 50 and 150 mg kg(-1)). Mineralization rates were estimated by quantification of 14CO2 emission from degradation of 14C-MCPA. tfdA mRNA was detected 1 h after amendment at all four concentrations. In soils amended with 2 and 20 mg kg(-1), the mRNA/DNA ratio for tfdA demonstrated a sharp transient maximum of tfdA expression from no to full expression within 3 and 6 h respectively, followed by a decline and complete loss of expression after 19 and 43 h. A more complex pattern of tfdA expression was observed for the higher 50 and 150 mg kg(-1) amendments; this coincided with growth of C. necator AEO106 (pRO101) in the system. Repeated amendment with MCPA after 2 weeks in the 20 mg kg(-1) scenario revealed a sharp increase of tfdA mRNA, and absence of a mineralization lag phase. For all amendments, tfdA mRNA was detectable only during active mineralization, and thus revealed a direct correlation between tfdA mRNA presence and microbial degrader activity. The present study demonstrates that direct analysis of functional gene expression dynamics by quantification of mRNA can indeed be made in natural soil.     

Mobilization of the genetically engineered plasmid pHSV106 from Escherichia coli HB101(pHSV106) to Enterobacter cloacae in drinking water.

We have used triparental matings to demonstrate transfer (mobilization) of the nonconjugative genetically engineered plasmid pHSV106, which contains the thymidine kinase gene of herpes simplex virus cloned into pBR322, from Escherichia coli HB101 to an environmental isolate of Enterobacter cloacae in sterile drinking water. This is the first demonstration of a two-step mobilization of a genetically engineered plasmid in any type of fresh water, including drinking water. Transfer was mediated by R plasmid R100-1 of E. coli ED2149(R100-1). Matings in drinking water at 15, 25, and 35 degrees C yielded recombinants, the number of which increased with increasing temperature. Numbers of recombinants obtained were 2 orders of magnitude lower than those obtained from matings in Trypticase soy broth. High concentrations of parental organisms (2.6 x 10(8) to 2.0 x 10(9) CFU/ml) were required. During 1 week of incubation in drinking water, number of parental organisms and recombinants resulting from mobilization remained constant in the absence of indigenous organisms and declined in their presence. Using oligonucleotide probes for the cloned foreign DNA (thymidine kinase gene) and plasmid vector DNA (ampicillin resistance gene), we demonstrated that both genes were transferred to E. cloacae in the mobilization process. In one recombinant selected for detailed study, the plasmids containing these genes differed in size from all forms of pHSV106 present in E. coli HB101(pHSV106), indicating that DNA rearrangement had occurred. This recombinant maintained its plasmids in unchanged form for 15 days in drinking water. A second rearrangement occurred during serial passage of this recombinant on selective media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mobilization of the genetically engineered plasmid pHSV106 from Escherichia coli HB101(pHSV106) to Enterobacter cloacae in drinking water

We have used triparental matings to demonstrate transfer (mobilization) of the nonconjugative genetically engineered plasmid pHSV106, which contains the thymidine kinase gene of herpes simplex virus cloned into pBR322, from Escherichia coli HB101 to an environmental isolate of Enterobacter cloacae in sterile drinking water. This is the first demonstration of a two-step mobilization of a genetically engineered plasmid in any type of fresh water, including drinking water. Transfer was mediated by R plasmid R100-1 of E. coli ED2149(R100-1). Matings in drinking water at 15, 25, and 35 degrees C yielded recombinants, the number of which increased with increasing temperature. Numbers of recombinants obtained were 2 orders of magnitude lower than those obtained from matings in Trypticase soy broth. High concentrations of parental organisms (2.6 x 10(8) to 2.0 x 10(9) CFU/ml) were required. During 1 week of incubation in drinking water, number of parental organisms and recombinants resulting from mobilization remained constant in the absence of indigenous organisms and declined in their presence. Using oligonucleotide probes for the cloned foreign DNA (thymidine kinase gene) and plasmid vector DNA (ampicillin resistance gene), we demonstrated that both genes were transferred to E. cloacae in the mobilization process. In one recombinant selected for detailed study, the plasmids containing these genes differed in size from all forms of pHSV106 present in E. coli HB101(pHSV106), indicating that DNA rearrangement had occurred. This recombinant maintained its plasmids in unchanged form for 15 days in drinking water. A second rearrangement occurred during serial passage of this recombinant on selective media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and survival of Pseudomonas cepacia DBO1(pRO101) in soil amended with 2,4-dichlorophenoxyacetic acid

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading pseudomonad, Pseudomonas cepacia DBO1(pRO101), was inoculated at approximately 10⁷ CFU/g into sterile and non-sterile soil amended with 0, 5 or 500 ppm 2,4-D and the survival of the strain was studied for a period of 44 days. In general, the strain survived best in sterile soil. When the sterile soil was amended with 2,4-D, the strain survived at a significantly higher level than in non-amended sterile soil. In non-sterile soil either non-amended or amended with 5 ppm 2,4-D the strain died out, whereas with 500 ppm 2,4-D the strain only declined one order of magnitude through the 44 days.The influence of 0,0.06, 12 and 600 ppm 2,4-D on short-term (48 h) survival of P. cepacia DBO1(pRO101) inoculated to a level of 6×10⁴, 6×10⁶ or 1×10⁸ CFU/g soil was studied in non-sterile soil. Both inoculum level and 2,4-D concentration were found to have a positive influence on numbers of P. cepacia DBO1(pRO101). At 600 ppm 2,4-D growth was significant irrespective of the inoculation level, and at 12 ppm growth was stimulated at the two lowest inocula levels. P. cepacia DBO1(pRO101) was able to survive for 15 months in sterile buffers kept at room temperature. During this starvation, cells shrunk to about one third the volume of exponentially growing cells.Abbreviations AODC acridine orange direct count - CFU colony forming units - PTYG-Agar peptone, tryptone, yeast & glucose agar - TET tetracycline - LB Luria Bertani medium     

Identification and molecular characterization of the acetyl coenzyme A synthetase gene (acoE) of Alcaligenes eutrophus.

The gene locus acoE, which is involved in the utilization of acetoin in Alcaligenes eutrophus, was identified as the structural gene of an acetyl coenzyme A synthetase (acetate:coenzyme A ligase [AMP forming]; EC 6.2.1.1). This gene was localized on a 3.8-kbp SmaI-EcoRI subfragment of an 8.1-kbp EcoRI restriction fragment (fragment E) that was cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The 1,983 bp acoE gene encoded a protein with a relative molecular weight of 72,519, and it was preceded by a putative Shine-Dalgarno sequence. A comparison analysis of the amino acid sequence deduced from acoE revealed a high degree of homology to primary structures of acetyl coenzyme A synthetases from other sources (amounting to up to 50.5% identical amino acids). Tn5 insertions in two transposon-induced mutants of A. eutrophus, that were impaired in the catabolism of acetoin were mapped 481 and 1,159 bp downstream from the translational start codon of acoE. The expression of acoE in Escherichia coli led to the formation of an acyl coenzyme A synthetase that accepted acetate as the preferred substrate (100% relative activity) but also reacted with propionate (46%) and hydroxypropionate (87%); fatty acids consisting of four or more carbon atoms were not accepted. In addition, evidence for the presence of a second acyl coenzyme A synthetase was obtained; this enzyme exhibited a different substrate specificity. The latter enzyme is obviously required for the activation of propionate, e.g., during the formation of the storage compound poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) when propionate is provided as the sole carbon source. An analysis of mutants provided evidence that the expression of the uptake protein for propionate depends on the presence of alternate sigma factor sigma 54.     

Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC)

The phbC gene encoding the third enzyme of the poly-beta-hydroxybutyrate biosynthetic pathway, poly-beta-hydroxybutyrate polymerase, in Alcaligenes eutrophus H16 has been identified by the complementation of poly-beta-hydroxybutyrate negative mutants of A. eutrophus H16. These results demonstrate that the three enzymes of the poly-beta-hydroxybutyrate biosynthetic pathway are organized phbC-phbA-phbB. Expression of all three genes in Escherichia coli results in a significant level (50% dry cell weight) of poly-beta-hydroxybutyrate production. phbC encodes a polypeptide of Mr = 63,900 which has a hydropathy profile distinct from typical membrane proteins indicating that poly-beta-hydroxybutyrate biosynthesis probably does not involve a membrane complex.     

Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli.

We studied recovery of poly(3-hydroxybutyric acid) (PHB) from Alcaligenes eutrophus and a recombinant Escherichia coli strain harboring the A. eutrophus poly(3-hydroxyalkanoic acid) biosynthesis genes. The amount of PHB degraded to a lower-molecular-weight compound in A. eutrophus during the recovery process was significant when sodium hypochlorite was used, but the amount degraded in the recombinant E. coli strain was negligible. However, there was no difference between the two microorganisms in the patterns of molecular weight change when PHB was recovered by using dispersions of a sodium hypochlorite solution and chloroform. To understand these findings, we examined purified PHB and lyophilized cells containing PHB by using a differential scanning calorimeter, a thermogravimetric analyzer, and nuclear magnetic resonance. The results of our analysis of lyophilized whole cells containing PHB with the differential scanning calorimeter suggested that the PHB granules in the recombinant E. coli strain were crystalline, while most of the PHB in A. eutrophus was in a mobile amorphous state. The stability of the native PHB in the recombinant E. coli strain during sodium hypochlorite treatment seemed to be due to its crystalline morphology. In addition, as determined by the thermogravimetric analyzer study, lyophilized cell powder of the recombinant E. coli strain containing PHB exhibited greater thermal stability than purified PHB obtained by chloroform extraction. The PHB preparations extracted from the two microorganisms had identical polymer properties.     

Plant protection and rhizosphere colonization of barley by seed inoculated herbicide degrading Burkholderia (Pseudomonas) cepacia DBO1(pRO101) in 2,4-D contaminated soil

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterium, Burkholderia cepacia (formerly Pseudomonas cepacia) DBO1(pRO101) was coated on non-sterile barley (Hordeum vulgare) seeds, which were planted in two non-sterile soils amended with varying amounts of 2,4-D herbicide. In the presence of 10 or 100 mg 2,4-D per kg soil B. cepacia DBO1(pRO101) readily colonized the root at densities up to 10⁷ CFU per cm root. In soil without 2,4-D the bacterium showed weak root colonization. The seeds coated with B. cepacia DBO1(pRO101) were able to germinate and grow in soils containing 10 or 100 mg kg^–1 2,4-D, while non-coated seeds either did not germinate or quickly withered after germination. The results suggest that colonization of the plant roots by the herbicide-degrading B. cepacia DBO1(pRO101) can protect the plant by degradation of the herbicide in the rhizosphere soil. The study shows that the ability to degrade certain pesticides should be considered, when searching for potential plant growth-promoting rhizobacteria. The role of root colonization by xenobiotic degrading bacteria is further discussed in relation to bioremediation of contaminated soils.     

Gene escape model: transfer of heavy metal resistance genes from Escherichia coli to Alcaligenes eutrophus on agar plates and in soil samples.

Conjugal transfer from Escherichia coli to Alcaligenes eutrophus of the A. eutrophus genes coding for plasmid-borne resistance to cadmium, cobalt, and zinc (czc genes) was investigated on agar plates and in soil samples. This czc fragment is not expressed in the donor strain, E. coli, but it is expressed in the recipient strain, A. eutrophus. Hence, expression of heavy metal resistance by cells plated on a medium containing heavy metals represents escape of the czc genes. The two plasmids into which this DNA fragment has been cloned previously and which were used in these experiments are the nonconjugative, mobilizable plasmid pDN705 and the nonconjugative, nonmobilizable plasmid pMOL149. In plate matings at 28 to 30 degrees C, the direct mobilization of pDN705 occurred at a frequency of 2.4 x 10(-2) per recipient, and the mobilization of the same plasmid by means of the IncP1 conjugative plasmids RP4 or pULB113 (present either in a third cell [triparental cross] or in the recipient strain itself [retromobilization]) occurred at average frequencies of 8 x 10(-4) and 2 x 10(-5) per recipient, respectively. The czc genes cloned into the Tra- Mob- plasmid pMOL149 were transferred at a frequency of 10(-7) to 10(-8) and only by means of plasmid pULB113. The direct mobilization of pDN705 was further investigated in sandy, sandy-loam, and clay soils. In sterile soils, transfer frequencies at 20 degrees C were highest in the sandy-loam soil (10(-5) per recipient) and were enhanced in all soils by the addition of easily metabolizable nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Turnover of poly(3-hydroxybutyrate) (PHB) and its influence on the molecular mass of the polymer accumulated by Alcaligenes eutrophus during batch culture

Abstract Radiolabelled glucose was added to a batch culture of Alcaligenes eutrophus during the accumulation of poly(3-hydroxybutyrate) (PHB) to label newly synthesized polymer. The specific radioactivity of the polymer continued to increase, by approximately 30%, after the cessation of PHB accumulation, indicating that turnover of PHB was occurring. Fractionation of PHB showed that high molecular mass polymer was gradually replaced by PHB of lower molecular mass. Turnover of PHB is the cause of the slow decline in the molecular mass of PHB following the cessation of polymer accumulation but is unlikely to be the sole reason for the more rapid decrease in the molecular mass of PHB during the accumulation phase.     

Inhibitor effects on the accumulation and efflux of nickel ions in plasmid pMOL28-harboring strains of Alcaligenes eutrophus

The accumulation and efflux of ⁶³Ni²⁺ ions were studied using two strains of the strictly respiratory bacterium Alcaligenes eutrophus, the wild type strain N9A and its transconjugant N9A-M243 which harbors plasmid pMOL28.1 encoding constitutive resistance to nickel. When 1 M ⁶³Ni²⁺ is added to respiring cells, strain N9A accumulates high, but M243 only negligibly small amounts of nickel. When the cells were preincubated for about 20 h under anoxic conditions and were then exposed to 1 M ⁶³Ni²⁺ anaerobically, both strains accumulated approximately the same amounts of nickel. Aeration of these preloaded cells resulted in rapid efflux by strain M243 but renewed uptake of nickel by N9A. ⁶³Ni²⁺ uptake and efflux are highly sensitive to protonophores such as CCCP, FCCP and TCS but insensitive to DCCD (each at 20 M concentrations). Measurements on the effects of the inhibitors on biosynthetic processes requiring ATP either from substrate phosphorylation or from electron transport phosphorylation made sure that at the concentrations used the inhibitor effects were specific. Thus the results suggest that in the nickelresistant strain M243 under normal aerobic conditions two constitutive energy-dependent nickel transport systems can function concomitantly, a chromosomally-determined specific uptake system and a plasmid-mediated specific nickel efflux system.     

Constitutive expression of the cloned phenol hydroxylase gene(s) from Alcaligenes eutrophus JMP134 and concomitant trichloroethylene oxidation.

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

Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli.

Eight mutants of Alcaligenes eutrophus defective in the intracellular accumulation of poly-beta-hydroxybutyric acid (PHB) were isolated after transposon Tn5 mutagenesis with the suicide vector pSUP5011. EcoRI fragments which harbor Tn5-mob were isolated from pHC79 cosmid gene banks. One of them, PPT1, was used as a probe to detect the intact 12.5-kilobase-pair EcoRI fragment PP1 in a lambda L47 gene bank of A. eutrophus genomic DNA. In six of these mutants (PSI, API, GPI, GPIV, GPV, and GPVI) the insertion of Tn5-mob was physically mapped within a region of approximately 1.2 kilobase pairs in PP1; in mutant API, cointegration of vector DNA has occurred. In two other mutants (GPII and GPIII), most probably only the insertion element had inserted into PP1. All PHB-negative mutants were completely impaired in the formation of active PHB synthase, which was measured by a radiometric assay. In addition, activities of beta-ketothiolase and of NADPH-dependent acetoacetyl coenzyme A (acetoacetyl-CoA) reductase were diminished, whereas the activity of NADPH-dependent acetoacetyl-CoA reductase was unaffected. In all PHB-negative mutants the ability to accumulate PHB was restored upon complementation in trans with PP1. The PHB-synthetic pathway of A. eutrophus was heterologously expressed in Escherichia coli. Recombinant strains of E. coli JM83 and K-12, which harbor pUC9-1::PP1, pSUP202::PP1, or pVK101::PP1, accumulated PHB up to 30% of the cellular dry weight. Crude extracts of these cells had significant activities of the enzymes PHB synthase, beta-ketothiolase, and NADPH-dependent acetoacetyl-CoA reductase. Therefore, PP1 most probably encodes all three genes of the PHB-synthetic pathway in A. eutrophus. In addition to PHB-negative mutants, we isolated mutants which accumulate PHB at a much lower rate than the wild type does. These PHB-leaky mutants exhibited activities of all three PHB-synthetic enzymes; Tn5-mob had not inserted into PP1, and the phenotype of the wild type could not be restored with fragment PP1. The rationale for this mutant type remains unknown.     

Gene escape model: transfer of heavy metal resistance genes from Escherichia coli to Alcaligenes eutrophus on agar plates and in soil samples

Conjugal transfer from Escherichia coli to Alcaligenes eutrophus of the A. eutrophus genes coding for plasmid-borne resistance to cadmium, cobalt, and zinc (czc genes) was investigated on agar plates and in soil samples. This czc fragment is not expressed in the donor strain, E. coli, but it is expressed in the recipient strain, A. eutrophus. Hence, expression of heavy metal resistance by cells plated on a medium containing heavy metals represents escape of the czc genes. The two plasmids into which this DNA fragment has been cloned previously and which were used in these experiments are the nonconjugative, mobilizable plasmid pDN705 and the nonconjugative, nonmobilizable plasmid pMOL149. In plate matings at 28 to 30 degrees C, the direct mobilization of pDN705 occurred at a frequency of 2.4 x 10(-2) per recipient, and the mobilization of the same plasmid by means of the IncP1 conjugative plasmids RP4 or pULB113 (present either in a third cell [triparental cross] or in the recipient strain itself [retromobilization]) occurred at average frequencies of 8 x 10(-4) and 2 x 10(-5) per recipient, respectively. The czc genes cloned into the Tra- Mob- plasmid pMOL149 were transferred at a frequency of 10(-7) to 10(-8) and only by means of plasmid pULB113. The direct mobilization of pDN705 was further investigated in sandy, sandy-loam, and clay soils. In sterile soils, transfer frequencies at 20 degrees C were highest in the sandy-loam soil (10(-5) per recipient) and were enhanced in all soils by the addition of easily metabolizable nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of the RpoS (KatF) sigma factor on maintenance of viability and culturability of Escherichia coli and Salmonella typhimurium in seawater.

The sigma factor RpoS is essential for stationary-phase-specific, multiple-stress resistance. We compared the viabilities (direct viable counts) and culturabilities (colony counts) in seawater of Escherichia coli and Salmonella typhimurium strains and those in which rpoS was deleted or which were deficient in guanosine 3',5'-bispyrophosphate (ppGpp) synthesis (relA spoT). RpoS, possibly via ppGpp regulation, positively influenced the culturability of these bacteria in oligotrophic seawater. This influence closely depended, however, upon the growth state of the cells and the conditions under which they were grown prior to their transfer to seawater. The protective effect of RpoS was observed only in stationary-phase cells grown at low osmolarity. A previous exposure of cells to high osmolarity (0.5 M NaCl) also had a strong influence on the effect of RpoS on cell culturability in seawater. Both E. coli and S. typhimurium RpoS mutants lost the ability to acquire a high resistance to seawater, as observed in both logarithmic-phase and stationary-phase RpoS+ cells grown at high osmolarity. A previous growth of S. typhimurium cells under anoxic conditions also modulated the incidence of RpoS on their culturability. When grown anaerobically at high osmolarity, logarithmic-phase S. typhimurium RpoS+ cells partly lost their resistance to seawater through preadaptation to high osmolarity. When grown anaerobically at high osmolarity until stationary phase, both RpoS+ and RpoS- cells retained very high levels of both viability and culturability and then did not enter the viable but nonculturable state for over 8 days in seawater because of an RpoS-independent, unknown mechanism.     

Catechol dioxygenases from Escherichia coli (MhpB) and Alcaligenes eutrophus (MpcI): sequence analysis and biochemical properties of a third family of extradiol dioxygenases.

The nucleotide sequence of the Escherichia coli mhpB gene, encoding 2,3-dihydroxyphenylpropionate 1,2-dioxygenase, was determined by sequencing of a 3.1-kb fragment of DNA from Kohara phage 139. The inferred amino acid sequence showed 58% sequence identity with the sequence of an extradiol dioxygenase, MpcI, from Alcaligenes eutrophus and 10 to 20% sequence identity with protocatechuate 4,5-dioxygenase from Pseudomonas paucimobilis, with 3,4-dihydroxyphenylacetate 2,3-dioxygenase from E. coli, and with human 3-hydroxyanthranilate dioxygenase. Sequence similarity between the N- and C-terminal halves of this new family of dioxygenases was detected, with conserved histidine residues in the N-terminal domain. A model is proposed to account for the relationship between this family of enzymes and other extradiol dioxygenases. The A. eutrophus MpcI enzyme was expressed in E. coli, purified, and characterized as a protein with a subunit size of 33.8 kDa. Purified MhpB and MpcI showed similar substrate specificities for a range of 3-substituted catechols, and evidence for essential histidine and cysteine residues in both enzymes was obtained.     

Further studies on RpoS in enterobacteria: identification of rpoS in Enterobacter cloacae and Kluyvera cryocrescens

RpoS, the alternative sigma factor sigma(s), is important for bacterial survival under extreme conditions. Many enterobacteria are opportunistic human pathogens and their ability to survive in a changing environment could be an essential step for their virulence. To determine the presence of this gene in enteric bacteria, an Escherichia coli rpoS probe was constructed and used to detect the presence of this gene in different species. A gene homologous to rpoS was found in Citrobacter amalonaticus, Enterobacter cloacae, Klebsiella planticola, Kluyvera cryocrescens, Serratia rubidaea, Shigella sonnei, and Yersinia ruckeri. Providencia stuartii and Proteus vulgaris were the only tested enterobacteria that did not show any signal with the E. coli rpoS probe or that did not lead to amplification of an rpoS fragment using specific primers. The rpoS gene from E. cloacae and from K. cryocrescens was cloned and sequenced and a mutant allele was constructed in E. cloacae. Survival rates under different harsh conditions were followed in order to determine the effect of rpoS inactivation in exponential- and stationary-phase cells of both strains. E. cloacae rpoS mutants were more sensitive to extreme pH, high osmolarity, and high temperature than the wild-type.     

Chromosome transfer and R-prime plasmid formation mediated by plasmid pULB113 (RP4::mini-Mu) in Alcaligenes eutrophus CH34 and Pseudomonas fluorescens 6.2.

Plasmid pULB113 (RP4::mini-Mu), which contains the mini-Mu transposon, promoted both homologous and heterologous gene transfer from Pseudomonas fluorescens 6.2 and Alcaligenes eutrophus CH34. Homologous gene transfer in P. fluorescens 6.2 and A. eutrophus CH34 occurred at a frequency of 10(-4) to 10(-5), and recombinants inherited unselected recessive markers, suggesting a process of chromosome mobilization. Loci involved in autotrophic growth were among those transferred in A. eutrophus. In heterospecific matings, markers were transferred from P. fluorescens to A. eutrophus, Salmonella typhimurium LT2, and Escherichia coli, from A. eutrophus to P. fluorescens, and from Erwinia carotovora subsp. chrysanthemi to A. eutrophus. Heterospecific matings resulted in the formation of R-prime plasmids at frequencies of 10(-7) to 10(-4) per transferred plasmid. When S. typhimurium was the recipient, we observed R-prime plasmids with both restriction-proficient and restriction-deficient strains, although restriction markedly affected the frequency of transfer of pULB113. R-prime plasmids were quite stable, but lost the transposed marker more easily in a rec+ background than in a recA background, suggesting excision of transposed material by reciprocal recombination between flanking copies of mini-Mu. R-prime plasmids could be transferred easily into different recipients and were used in complementation studies. PstI restriction digests of four R-prime plasmids carrying P. fluorescens 6.2 DNA showed a number of additional bands, suggesting that several genes were transposed together with the selected marker on the plasmid.