Deletion of the parA (soj) homologue in Pseudomonas aeruginosa causes ParB instability and affects growth rate, chromosome segregation, and motility

The parA and parB genes of Pseudomonas aeruginosa are located approximately 8 kb anticlockwise from oriC. ParA is a cytosolic protein present at a level of around 600 molecules per cell in exponential phase, but the level drops about fivefold in stationary phase. Overproduction of full-length ParA or the N-terminal 85 amino acids severely inhibits growth of P. aeruginosa and P. putida. Both inactivation of parA and overexpression of parA in trans in P. aeruginosa also lead to accumulation of anucleate cells and changes in motility. Inactivation of parA also increases the turnover rate (degradation) of ParB. This may provide a mechanism for controlling the level of ParB in response to the growth rate and expression of the parAB operon.     

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.     

Cell density-dependent gene contributes to efficient seed colonization by Pseudomonas putida KT2440

We have characterized the expression pattern of a gene, ddcA, involved in initial colonization of corn seeds by Pseudomonas putida KT2440. The ddcA gene codes for a putative membrane polypeptide belonging to a family of conserved proteins of unknown function. Members of this family are widespread among prokaryotes and include the products of a Salmonella enterica serovar Typhimurium gene expressed during invasion of macrophages and psiE, an Escherichia coli phosphate starvation-inducible gene. Although its specific role is undetermined, the presence of ddcA in multicopy restored the seed adhesion capacity of a KT2440 ddcA mutant. Expression of ddcA is growth phase regulated, being maximal at the beginning of stationary phase. It is independent of RpoS, nutrient depletion, or phosphate starvation, and it is not the result of changes in the medium pH during growth. Expression of ddcA is directly dependent on cell density, being also stimulated by the addition of conditioned medium and of seed exudates. This is the first evidence suggesting the existence of a quorum-sensing system in P. putida KT2440. The potential implication of such a signaling process in seed adhesion and colonization by the bacterium is discussed.     

Minicell-forming mutants of Escherichia coli: production of minicells and anucleate rods.

The Escherichia coli minB mutant originally isolated is known to septate at cell poles to form spherical anucleate minicells. Three new minicell-producing mutants were isolated during a screening by autoradiography for chromosome partition mutants giving rise spontaneously to normal-sized anucleate cells. These min mutants were affected close to or in the minB locus. Autoradiography analysis as well as fluorescent staining of DNA showed that in addition to minicells, these strains and the original minB mutant also spontaneously produced anucleate rods of normal size and had an abnormal DNA distribution in filaments. These aberrations were not associated with spontaneous induction of the SOS response. Inhibition of DNA synthesis in these mutants gave rise to anucleate cells whose size was longer than unit cell length, suggesting that the min defect allows septation to take place at normally forbidden sites not only at cell poles but also far from poles. Abnormal DNA distribution and production of anucleate rods suggest that the Min product(s) could be involved in DNA distribution.     

Distinct centromere-like parS sites on the two chromosomes of Vibrio spp

Vibrio cholerae, the cause of cholera, has two circular chromosomes. The parAB genes on each V. cholerae chromosome act to control chromosome segregation in a replicon-specific fashion. The chromosome I (ChrI) parAB genes (parAB1) govern the localization of the origin region of ChrI, while the chromosome II (ChrII) parAB genes (parAB2) control the segregation of ChrII. In addition to ParA and ParB proteins, Par systems require ParB binding sites (parS). Here we identified the parS sites on both V. cholerae chromosomes. We found three clustered origin-proximal ParB1 binding parS1 sites on ChrI. Deletion of these three parS1 sites abrogated yellow fluorescent protein (YFP)-ParB1 focus formation in vivo and resulted in mislocalization of the ChrI origin region. However, as observed in a parA1 mutant, mislocalization of the ChrI origin region in the parS1 mutant did not compromise V. cholerae growth, suggesting that additional (non-Par-related) mechanisms may mediate the partitioning of ChrI. We also identified 10 ParB2 binding parS2 sites, which differed in sequence from parS1. Fluorescent derivatives of ParB1 and ParB2 formed foci only with the cognate parS sequence. parABS2 appears to form a functional partitioning system, as we found that parABS2 was sufficient to stabilize an ordinarily unstable plasmid in Escherichia coli. Most parS2 sites were located within 70 kb of the ChrII origin of replication, but one parS2 site was found in the terminus region of ChrI. In contrast, in other sequenced vibrio species, the distribution of parS1 and parS2 sites was entirely chromosome specific.     

Isolation and characterization of solvent-tolerant Pseudomonas putida strain T-57, and its application to biotransformation of toluene to cresol in a two-phase (organic-aqueous) system

Pseudomonas putida T-57 was isolated from an activated sludge sample after enrichment on mineral salts basal medium with toluene as a sole source of carbon. P. putida T-57 utilizes n-butanol, toluene, styrene, m-xylene, ethylbenzene, n-hexane, and propylbenzene as growth substrates. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10-90% (v/v), and was tolerant to organic solvents whose log P(ow) (1-octanol/water partition coefficient) was higher than 2.5. Enzymatic and genetic analysis revealed that P. putida T-57 used the toluene dioxygenase pathway to catabolize toluene. A cis-toluene dihydrodiol dehydrogenase gene (todD) mutant of T-57 was constructed using a gene replacement technique. The todD mutant accumulated o-cresol (maximum 1.7 g/L in the aqueous phase) when cultivated in minimal salts basal medium supplemented with 3% (v/v) toluene and 7% (v/v) 1-octanol. Thus, T-57 is thought to be a good candidate host strain for bioconversion of hydrophobic substrates in two-phase (organic-aqueous) systems.     

Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.     

Partition of P1 plasmids in Escherichia coli mukB chromosomal partition mutants.

The partition system of the low-copy-number plasmid/prophage of bacteriophage P1 encodes two proteins, ParA and ParB, and contains a DNA site called parS. ParB and the Escherichia coli protein IHF bind to parS to form the partition complex, in which parS is wrapped around ParB and IHF in a precise three-dimensional conformation. Partition can be thought of as a positioning reaction; the plasmid-encoded components ensure that at least one copy of the plasmid is positioned within each new daughter cell. We have used an E. coli chromosomal partition mutant to test whether this positioning is mediated by direct plasmid-chromosomal attachment, for example, by pairing of the partition complex that forms at parS with a bacterial attachment site. The E. coli MukB protein is required for proper chromosomal positioning, so that mukB mutants generate some cells without chromosomes (anucleate cells) at each cell division. We analyzed the plasmid distribution in nucleate and anucleate mukB cells. We found that P1 plasmids are stable in mukB mutants and that they partition into both nucleate and anucleate cells. This indicates that the P1 partition complex is not used to pair plasmids with the host chromosome and that P1 plasmids must be responsible for their own proper cellular localization, presumably through host-plasmid protein-protein interactions.     

A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition

We have analysed the function of a gene of Bacillus subtilis , the product of which shows significant homology with eukaryotic SMC proteins essential for chromosome condensation and segregation. Two mutant strains were constructed; in one, the expression was under the control of the inducible spac promoter (conditional null) and, in the other, the gene was disrupted by insertion (disrupted null). Both could form colonies at 23°C but not at 37°C in the absence of the expression of the Smc protein, indicating that the B. subtilis smc gene was essential for cell growth at higher temperatures. Microscopic examination revealed the formation of anucleate and elongated cells and diffusion of nucleoids within the elongated cells in the disrupted null mutant grown at 23°C and in the conditional null mutant grown in low concentrations of IPTG at 37°C. In addition, immunofluorescence microscopy showed that subcellular localization of the Spo0J partition protein was irregular in the smc disrupted null mutant, compared with bipolar localization in wild-type cells. These results indicate that the B. subtilis smc gene is essential for chromosome partition. The role of B. subtilis Smc protein in chromosome partition is discussed.     

Chromosome partitioning in Escherichia coli: novel mutants producing anucleate cells.

To study the chromosomal partitioning mechanism in cell division, we have isolated a novel type of Escherichia coli mutants which formed anucleate cells, by using newly developed techniques. One of them, named mukA1, is not lethal and produces normal-sized anucleate cells at a frequency of 0.5 to 3% of total cells in exponentially growing populations but does not produce filamentous cells. Results suggest that the mutant is defective in the chromosome positioning at regular intracellular positions and fails frequently to partition the replicated daughter chromosomes into both daughter cells, resulting in production of one anucleate daughter cell and one with two chromosomes. The mukA1 mutation causes pleiotropic effects: slow growth, hypersensitivity to sodium dodecyl sulfate, and tolerance to colicin E1 protein, in addition to anucleate cell formation. Cloning of the mukA gene indicates that the mukA1 mutation is recessive and that the mukA gene is identical to the tolC gene coding for an outer membrane protein.     

ColRS two-component system prevents lysis of subpopulation of glucose-grown Pseudomonas putida

ColRS two-component system is well conserved in pseudomonads, but its exact role has remained obscure. Here, we report that Pseudomonas putida deficient in ColR experiences serious carbon source-specific stress that leads to the lysis of a subpopulation of bacteria growing on solid glucose medium. We observed that on glucose medium colR-deficient bacteria aggregated, produced a Congo Red-binding substance and had enhanced membrane permeability. Detection of a large amount of cytoplasmic beta-galactosidase and other proteins as well as chromosomal DNA in the growth medium of a colR mutant indicated that cell lysis took place if ColR was absent. Investigation of colony morphology revealed concavities in the centre of the colonies of colR mutant suggesting that cell lysis occurred mainly in the areas of the highest cell density. Analysis of bacteria at a single cell level by flow cytometry showed that population of glucose-grown colR-deficient cells was heterogeneous. In addition to the wild type-like population, we detected a subpopulation of cells with damaged membrane permeable to propidium iodide. Interestingly, inactivation of oprB1 encoding a glucose porin eliminated the cell lysis as well as autoaggregation and membrane leakiness of a colR mutant indicating that glucose influx could be responsible for membrane stress in the absence of ColRS system.     

Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida

The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase. A 500-bp 'cyoB' DNA fragment was cloned in pCHESI Omega Km and used to generate a cyoB knock-out mutant in vivo. The mutant strain was not limited in the generation of proton-motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild-type strain. This effect was even more pronounced when toluene was supplied in the gas phase. In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild-type strain. This effect was particularly exacerbated in cells that reached the stationary phase. The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent-induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.     

Sigma 54 levels and physiological control of the Pseudomonas putida Pu promoter

The cellular levels of the alternative sigma factor sigma(54) of Pseudomonas putida have been examined in a variety of growth stages and culture conditions with a single-chain Fv antibody tailored for detection of scarce proteins. The levels of sigma(54) were also monitored in P. putida strains with knockout mutations in ptsO or ptsN, known to be required for the C-source control of the sigma(54)-dependent Pu promoter of the TOL plasmid. Our results show that approximately 80 +/- 26 molecules of sigma(54) exist per cell. Unlike that in relatives of Pseudomonas (e.g., Caulobacter), where fluctuations of sigma(54) determine adaptation and differentiation when cells face starvation, sigma(54) in P. putida remains unexpectedly constant at different growth stages, in nitrogen starvation and C-source repression conditions, and in the ptsO and ptsN mutant strains analyzed. The number of sigma(54) molecules per cell in P. putida is barely above the predicted number of sigma(54)-dependent promoters. These figures impose a framework on the mechanism by which Pu (and other sigma(54)-dependent systems) may become amenable to physiological control.     

Anucleate cell production and surface extension in a temperature-sensitive chromosome initiation mutant of Bacillus subtilis.

At 45 C, in a temperature-sensitive initiation mutant (TsB134) of Bacillus subtilis 168 Thy- tryp-, growing in a glucose-arginine minimal medium, chromosome completion occurred over a period of 80 to 90 min, after which there was no further nuclear division. Normal symmetrical cell divisions continued for a generation afterwards, so that nuclei were segregated into separate cells. During this period asymmetric divisions started to occur. Septa appeared at 25 to 30% from one end of the cell, giving a small anucleate cell and a larger nucleate cell. During inhibition of deoxyribonucleic acid (DNA) synthesis by thymine starvation under the restrictive conditions, asymmetrical division also occurred until there was approximately one nucleus per cell (about one generation time). Asymmetric division, giving anucleate cells, then occurred. Similar results were obtained when DNA synthesis was inhibited by nalidixic acid. After 3 h at 45 C, the rate of anucleate cell production in the presence and absence of thymine was constant at one division per 85 min per chromosome terminus present when DNA synthesis stopped. In the absence of DNA synthesis (during thymine starvation) at 35 C, growth in cell length was linear (i.e., the rate was constant), but at 45 C during thymine starvation the rate gradually increased by more than twofold. It is suggested that this was due to the establishment of new sites of growth associated with anucleate cell production. In the presence of thymine at 45 C, the rate of length extension increased by more than fourfold, which it is suggested was caused by the appearance of new growth zones as a result of chromosome termination and a contribution associated with anucleate cell production. If the mutant was incubated at 45 C for 90 min, both in the presence and absence of thymine, then anucleate cell formation could continue on restoration to 35 C in the absence of thymine...     

Biofilm formation of Pseudomonas putida IsoF: the role of quorum sensing as assessed by proteomics

Pseudomonas putida strains are frequently isolated from the rhizosphere of plants and many strains promote plant-growth, exhibit antagonistic activities against plant pathogens and have the capacity to degrade pollutants. Factors that appear to contribute to the rhizosphere fitness are the ability of the organism to form biofilms and the utilization of cell-to-cell-communication systems (quorum sensing, QS) to co-ordinate the expression of certain phenotypes in a cell density dependent manner. Recently, the ppu QS locus of the tomato rhizosphere isolate P. putida Iso F was characterized and an isogenic QS-negative ppuI mutant P. putida F117 was generated. In the present study we investigated the impact of QS and biofilm formation on the protein profile of surface-associated proteins of P. putida IsoF. This was accomplished by comparative proteome analyses of the P. putida wild type IsoF and the QS-deficient mutant F117 grown either in planktonic cultures or in 60 h old mature biofilms. Differentially expressed proteins were identified by peptide mass fingerprinting and database search in the completed P. putida KT2440 genome sequence. The sessile life style affected 129 out of 496 surface proteins, suggesting that a significant fraction of the bacterial genome is involved in biofilm physiology. In surface-attached cells 53 out of 484 protein spots were controlled by the QS system, emphasizing its importance as global regulator of gene expression in P. putida IsoF. Most interestingly, the impact of QS was dependent on whether cells were grown on a surface or in suspension; about 50% of the QS-controlled proteins identified in planktonic cultures were found to be oppositely regulated when the cells were grown as biofilms. Fifty-seven percent of all identified surface-controlled proteins were also regulated by the ppu QS system. In conclusion, our data provide strong evidence that the set of QS-regulated proteins overlaps substantially with the set of proteins differentially expressed in sessile cells.     

Characteristic of mutants of Pseudomonas putida IPM-36 recombinant strains, selected by anticipating growth on a media containing an inducer of cry3A gene expression

Induction of the expression of the delta-endotoxin gene from Bacillus thuringiensis var. tenebrionis in the recombinant strain Pseudomonas putida IPM-36 negatively affected the viability and the growth rate of the culture. In order to optimize the insecticide production by the recombinant strain, mutant clones exhibiting anticipating growth on an inducer-containing medium were selected and studied. These clones differed in such aspects as the localization of mutations (either in plasmid pBTN11, carrying the cry3A gene, or in the chromosome), growth rate, or the level of delta-endotoxin synthesis after induction. Several mutants obtained proved much superior to P. putida IPM-36 in their structural and segregation stability, although they were as efficient as the original strain with respect to the production of the insecticide (protei Cry3A).     

Superoxide dismutase activity in Pseudomonas putida affects utilization of sugars and growth on root surfaces

To investigate the role of superoxide dismutases (SOD) in root colonization and oxidative stress, mutants of Pseudomonas putida lacking manganese-superoxide dismutase (MnSOD) (sodA), iron-superoxide dismutase (FeSOD) (sodB), or both were generated. The sodA sodB mutant did not grow on components washed from bean root surfaces or glucose in minimal medium. The sodB and sodA sodB mutants were more sensitive than wild type to oxidative stress generated within the cell by paraquat treatment. In single inoculation of SOD mutants on bean, only the sodA sodB double mutant was impaired in growth on root surfaces. In mixed inoculations with wild type, populations of the sodA mutant were equal to those of the wild type, but levels of the sodB mutant and, to a great extent, the sodA sodB mutant, were reduced. Confocal microscopy of young bean roots inoculated with green fluorescent protein-tagged cells showed that wild type and SOD single mutants colonized well predominantly at the root tip but that the sodA sodB double mutant grew poorly at the tip. Our results indicate that FeSOD in P. putida is more important than MnSOD in aerobic metabolism and oxidative stress. Inhibition of key metabolic enzymes by increased levels of superoxide anion may cause the impaired growth of SOD mutants in vitro and in planta.     

Global features of the Pseudomonas putida KT2440 genome sequence

The compositional bias of the G+C, di- and tetranucleotide contents in the 6 181 862 bp Pseudomonas putida KT2440 genome was analysed in sliding windows of 4000 bp in steps of 1000 bp. The genome has a low GC skew (mean 0.066) between the leading and lagging strand. The values of GC contents (mean 61.6%) and of dinucleotide relative abundance exhibit skewed Gaussian distributions. The variance of tetranucleotide frequencies, which increases linearly with increasing GC content, shows two overlapping Gaussian distributions of genome sections with low (minor fraction) or high variance (major fraction). Eighty per cent of the chromosome shares similar GC contents and oligonucleotide bias, but 105 islands of 4000 bp or more show atypical GC contents and/or oligonucleotide signature. Almost all islands provide added value to the metabolic proficiency of P. putida as a saprophytic omnivore. Major features are the uptake and degradation of organic chemicals, ion transport and the synthesis and secretion of secondary metabolites. Other islands endow P. putida with determinants of resistance and defenceor with constituents and appendages of the cell wall. A total of 29 islands carry the signature of mobile elements such as phage, transposons, insertion sequence (IS) elements and group II introns, indicating recent acquisition by horizontal gene transfer. The largest gene carries the most unusual sequence that encodes a multirepeat threonine-rich surface adhesion protein. Among the housekeeping genes, only genes of the translational apparatus were located in segments with an atypical signature, suggesting that the synthesis of ribosomal proteins is uncoupled from the rapidly changing translational demands of the cell by the separate utilization of tRNA pools.