The consensus sequence of ice nucleation proteins from Erwinia herbicola, Pseudomonas fluorescens and Pseudomonas syringae

The consensus sequence of three bacterial ice nucleation proteins was determined by extrapolation from the nucleotide (nt) sequences of three ice nucleation-encoding genes, iceE (presented here), inaW and inaZ. The three proteins possess considerable similarity, so that a preferred amino acid is shown in most positions of the consensus. The corresponding genes show considerable divergence in the third nt positions of synonymous codons, suggesting that the proteins' conserved features have been maintained by selection. Therefore, the consensus sequence is likely to represent the components of primary structure most important to the ice nucleation function.     

Inhibition by sulfide of nitric and nitrous oxide reduction by denitrifying Pseudomonas fluorescens.

The influence of low redox potentials and H2S on NO and N2O reduction by resting cells of denitrifying Pseudomonas fluorescens was studied. Hydrogen sulfide and Ti(III) were added to achieve redox potentials near -200 mV. The control without reductant had a redox potential near +200 mV. Production of 13NO, [13N]N2O, and [13N]N2 from 13NO3- and 13NO2- was followed. Total gas production was similar for all three treatments. The accumulation of 13NO was most significant in the presence of sulfide. A parallel control with autoclaved cells indicated that the 13NO production was largely biological. The sulfide inhibition was more dramatic at the level of N2O reduction; [13N]N2O became the major product instead of [13N]N2, the dominant product when either no reductant or Ti(III) was present. The results indicate that the specific action of sulfide rather than the low redox potential caused a partial inhibition of NO reduction and a strong inhibition of N2O reduction in denitrifying cells.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

The Effect of Water Activity on the Growth and Respiration of Pseudomonas fluorescens

The growth rate of Pseudomonas fluorescens was greater and continued at lower water activity ( a _w) values when glycerol controlled the a _w of glucose minimal medium than when the a _w was controlled by NaCl and sucrose. Growth was not observed below 0·945, 0·970 and 0·964 a _w when glycerol, sucrose and NaCl respectively controlled the a _w. The catabolism of glucose, Na lactate and DL-arginine as measured by respirometry was completely inhibited at a _w values greater than the minimum for growth when the a _w was controlled with NaCl. When the a _w was controlled with glycerol, catabolism of the three substrates continued at a _w values significantly below the a _w for growth on glucose. Catabolism of glucose in the presence of sucrose occurred at a level below the minimum growth a _w but catabolism of the other two substrates ceased at a _w values greater than the minimum growth a _w. Arrhenius plots between 10° and 34°C of the growth rate in glucose minimal medium at 0·98 a _w showed that the order of inhibition was sucrose > NaCl > glycerol. The order of inhibition differed when Arrhenius plots of catabolism of glucose was examined between 10° and 34 °C, namely NaCl > sucrose > glycerol. The mechanism of action of solutes controlling a _w is discussed.     

Evaluation of Fleroxacin Activity against Established Pseudomonas fluorescens Biofilms

Scanning confocal laser microscopy (SCLM) and fluorescent molecular probes were used to evaluate the effect of the fluoroquinolone fleroxacin on the architecture of established Pseudomonas fluorescens biofilms. Control P. fluorescens biofilms were heterogeneous, consisting of cell aggregates extending from the attachment surface to maximum measured depths of ~90 μm (mean biofilm depth at 72 h, 42 ± 28 μm) and penetrated by an array of channels. In contrast, fleroxacin-treated biofilms were less deep (mean biofilm depth at 72 h, 29 ± 8 μm), varied little in depth over large areas, and consisted of a homogeneous distribution of cells. Fleroxacin also caused cells to elongate, with cells located near the biofilm-liquid interface lengthening significantly more than cells located at the attachment surface. By using SCLM, acridine orange, and image analysis it was found that ~59% of cells within fleroxacin-treated biofilms emitted red fluorescence whereas >99% of cells from control biofilms emitted green fluorescence. The fleroxacin-treated cells which emitted red fluorescence were observed to be the population of cells which elongated.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa.

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Immunological characterization of ice nucleation proteins from Pseudomonas syringae, Pseudomonas fluorescens, and Erwinia herbicola.

Antibodies were raised against the InaW protein, the product of the ice nucleation gene of Pseudomonas fluorescens MS1650, after protein isolation from an Escherichia coli clone. On Western blots (immunoblots), these antibodies recognized InaW protein and InaZ protein (the ice nucleation gene product of Pseudomonas syringae S203), produced by both E. coli clones and the source organisms. The InaZ protein appeared in P. syringae S203 during stationary phase; its appearance was correlated with the appearance of the ice nucleation-active phenotype. In contrast, the InaW protein occurred at relatively constant levels throughout the growth phases of P. fluorescens MS1650; the ice nucleation activity was also constant. Western analyses of membrane preparations of P. syringae PS31 and Erwinia herbicola MS3000 with this antibody revealed proteins which were synthesized with development of the nucleating phenotype. In these species the presence or absence of the nucleating phenotype was controlled by manipulation of culture conditions. In all nucleation-positive cultures examined, cross-reacting low-molecular-weight bands were observed; these bands appeared to be products of proteolytic degradation of ice nucleation proteins. The proteolysis pattern of InaZ protein seen on Western blots showed a periodic pattern of fragment sizes, suggesting a highly repetitive site for protease action. A periodic primary structure is predicted by the DNA sequence of the inaZ gene.     

The metabolism of halogen-substituted benzoic acids by Pseudomonas fluorescens

1. Washed suspensions of Pseudomonas fluorescens, grown with benzoate as sole carbon source, oxidize monohalogenobenzoates in the following descending order of effectiveness: benzoate, fluorobenzoates, chlorobenzoates, bromobenzoates, iodobenzoates. 2. Cells grown on asparagine oxidize benzoate after an adaptive period of 90–120min. This adaptive period is increased by halogenobenzoates in the following approximate descending order of effectiveness: chlorobenzoates, fluorobenzoates (=bromobenzoates), iodobenzoates. This inhibition of adaptation by halogeno analogues depends on the concentration of benzoate and is thus apparently competitive. 3. Cells do not adapt to oxidize the halobenzoates when the halogeno analogues are inducers. However, the fluorobenzoates reduce the lag period taken to form the benzoate-oxidizing system. 4. The halogenobenzoates inhibit adaptation to citrate and nicotinate but not so effectively as benzoate itself. This is presumably a `diauxic' effect. The analogues do not inhibit adaptation to catechol. 5. The halogenobenzoates are not used as sole carbon source for growth nor do they increase growth when cells grow with asparagine as the main carbon source. 6. It is suggested that this inability to use the analogues for growth is due partly to inability of the cells to liberate the halogen and to carry the oxidation to a stage at which carbon may be assimilated and partly to the inhibition of the induction of the oxidizing enzymes.     

Adaptation of Pseudomonas fluorescens to Low Levels of Water Activity Produced by Different Solutes

A culture of Pseudomonas fluorescens which grew at a minimal a_w of 0.97 in a glucose-salts broth adjusted to that level with NaCl, KCl, or glucose, and at a_w 0.9725 with Na₂SO₄, was adapted to grow at lower a_w levels produced by these solutes. Adaptation to growth at a low a_w produced by means of one solute did not necessarily result in growth at that a_w when another solute was employed to lower the a_w. The culture adapted to grow at a_w 0.9650 produced by addition of glucose could grow equally well in broth adjusted to that a_w with NaCl or KCl, but not Na₂SO₄. NaCl and KCl permitted growth at the lowest a_w level (0.9625), and the culture adapted in KCl did equally well in NaCl.     

Transport of maltose by Pseudomonas fluorescens W

The system for uptake of maltose in Pseudomonas fluorescens W was inducible. Using a mutant strain unable to hydrolyze maltose, it was shown that maltose was taken up unaltered against a concentration gradient. Uptake of ¹⁴C maltose was only significantly inhibited by nonradioactive maltose or maltotriose. These were the only sugars that could displace accumulated radioactive maltose in the strain unable to hydrolyze maltose. Uptake exhibited saturation kinetics and was inhibited by energy poisons, indicating that this system was one of active transport. Sulfhydryl-binding reagents reversibly inhibited maltose uptake. No transport ability was lost when cells were subjected to osmotic shock. Using the protein-binding dye 7-diazonium-1,3-naphthalene disulfonate a protein or proteins located in or external to the cell membrane was implicated in maltose transport. The hydrolysis of p-nitrophenyl--D-glucoside (PNPG) was used as an indirect measure of transport ability since penetration of PNPG, not its hydrolysis, was the rate-limiting step.Abbreviations PNPG paranitrophenyl--D-glucoside - NDS 7-diazonium-1,3-naphthalene disulfonic acid - PMB p-hydroxymercuribenzoate - MBS p-chloromercuriphenylsulfonic acid - PCMB p-chloromercuribenzoate - CCCP carbonyl cyanide m-chlorophenylhydrazone - DNP 2,4-dinitrophenol - HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide     

The effect of immobilization on rhamnolipid production by Pseudomonas fluorescens

Pseudomonas fluorescens in free suspension and immobilized to a commercially available biosupport (Biofix) and a biosorbent (Drizit), were used as bioremediation agents in an aqueous system with petrol (Slovene diesel) as the carbon source. Analysis of cellular growth and estimation of rhamnolipid production was carried out on the free suspension of the free and immobilized systems over 5 d. An increase in growth and rhamnolipid production was seen in the immobilized systems in comparison to the free system. EDTA was shown to be an inhibitor of rhamnolipid production. Its addition to the aqueous suspensions of all systems resulted in a fall in production of the surface-active agent in all cases, with no corresponding decrease in growth. This indicates the bacteria can rely on contact between the cell and the oil droplet for hydrocarbon transport into the cell. The data from this study indicated that immobilization resulted in a combination of increased contact between cell and hydrocarbon droplets and enhanced levels of rhamnolipid production.     

Nitrite inhibition of denitrification by Pseudomonas fluorescens

Using a pure culture of Pseudomonas fluorescens as a model system nitrite inhibition of denitrification was studies. A mineral media with acetate and nitrate as sole electron donor and acceptor, respectively, was used. Results obtained in continuous stirred-tank reactors (CSTR) operated at pH values between 6.6 and 7.8 showed that growth inhibition depended only on the nitrite undissociated fraction concentration (nitrous acid). A mathematical model to describe this dependence is put forward. The maximum nitrous acid concentration compatible with cell growth and denitrification activity was found to be 66 mug N/L. Denitrification activity was partially associated with growth, as described by the Luedeking-Piret equation. However, when the freshly inoculated reactor was operated discontinuosly, nitrite accumulation caused growth uncoupling from denitrification activity. The authors suggest that these results can be interpreted considering that (a) nitrous acid acts as a proton uncoupler; and (b) cultures continuoulsy exposed to nitrous acid prevent the uncoupling effect but not the growth inhibition. Examination of the growth dependence on nitrite concentration at pH 7.0 showed that adapted cultures (grown on CSTR) are less sensitive to nitrous acid inhibition than the ones cultivated in batch. (c) 1995 John Wiley & Sons, Inc.     

Fluorene cometabolism by Rhodococcus rhodochrous and Pseudomonas fluorescens

The transformation of fluorene by Rhodococcus rhodochrous strain 172 grown on sucrose and Pseudomonas fluorescens strain 26K grown on glycerol was studied as a function of the substrate concentration and the growth phase. Under certain cultivation conditions, fluorene was completely consumed from the medium. The specific transformation rate of fluorene was considerably higher when it was transformed in the presence of the cosubstrates than when it served as the sole carbon source. An approach to the evaluation of the specific transformation rate of fluorene during batch cultivations is proposed.