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. 2006 Jan;72(1):472–477. doi: 10.1128/AEM.72.1.472-477.2006

Involvement of Cyclopropane Fatty Acids in the Response of Pseudomonas putida KT2440 to Freeze-Drying

Jesús Muñoz-Rojas 1,†, Patricia Bernal 1,†, Estrella Duque 1, Patricia Godoy 1, Ana Segura 1, Juan-Luis Ramos 1,*
PMCID: PMC1352226  PMID: 16391080

Abstract

Pseudomonas putida KT2440, a saprophytic soil bacterium that colonizes the plant root, is a suitable microorganism for the removal of pollutants and a stable host for foreign genes used in biotransformation processes. Because of its potential use in agriculture and industry, we investigated the conditions for the optimal preservation of the strain and its derivatives for long-term storage. The highest survival rates were achieved with cells that had reached the stationary phase and which had been subjected to freeze-drying in the presence of disaccharides (trehalose, maltose, and lactose) as lyoprotectants. Using fluorescence polarization techniques, we show that cell membranes of KT2440 were more rigid in the stationary phase than in the exponential phase of growth. This is consistent with the fact that cells grown in the stationary phase exhibited a higher proportion of C17:cyclopropane as a fatty acid than cells in the exponential phase. Mutants for the cfaB gene, which encodes the main C17:cyclopropane synthase, and for the cfaA gene, which encodes a minor C17:cyclopropane synthase, were constructed. These mutants were more sensitive to freeze-drying than wild-type cells, particularly the mutant with a knockout in the cfaB gene that produced less than 2% of the amount of C17:cyclopropane produced by the parental strain.

Pseudomonas putida KT2440 is a well-characterized saprophytic member of the family Pseudomonaceae (38) certified by the National Institutes of Health as a secure host for foreign gene cloning (3). This microorganism uses a number of aromatic compounds, amino acids, and organic acids as carbon and energy sources (2, 34, 39), and it is able to adhere to abiotic and biotic surfaces, being an excellent colonizer of the plant roots (26). The complete genome sequence of P. putida KT2440 (27) reveals its nonpathogenic nature and also its potential for applications in agriculture, biocatalysis, bioremediation, and bioplastic production (19, 27, 30).

The availability of complete genome sequences facilitates research with genomics and proteomics, although many of these studies require a collection of mutants large enough to establish unequivocal gene and function relationships to identify regulons and to determine the hierarchy in transcriptional processes. We have constructed an ordered bank of mutants of P. putida (E. Duque [www.artemisa.eez.csic.es]) to facilitate detailed functional genomic studies of this microorganism. Long-term storage conservation of the mutant collection is of critical importance. Freezing at −80°C is frequently used for high-throughput analyses and to search for defined phenotypes. However, lyophilization is the most appropriate method for the long-term preservation of the clones. Lyophilization involves the use of lyoprotectants and myoinositol has been used with gram-negative bacteria in general (13, 29, 37) and with Pseudomonas in particular (E. Duque, unpublished results). However, upon lyophilization of P. putida KT2440, the recovery rate is on the order of 1 out of 104 original cells. To improve survival after lyophilization, we explored here the effect of growth conditions, growth phase, and the nature of lyoprotectants on the lyophilization of P. putida KT2440.

Our results indicated that survival of the strain was influenced by the bacterial growth phase and by the type and concentration of lyoprotectants used. We found that growth conditions that result in an increase in the relative amount of C17:cyclopropane fatty acid yielded better survival after lyophilization and that of the compounds we tested trehalose was the best lyoprotectant. We identified cfaB as the gene that encodes the major C17:cyclopropane synthase in KT2440 and generated a cfaB mutant which, in consonance with the low C17:cyclopropane levels, exhibited reduced survival rates after lyophilization.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The strains and plasmids used in the present study are listed in Table 1. Bacterial cells were grown at 30°C in a Kühner orbital shaker operated at 200 rpm in LB medium or M9 minimal medium containing 20 mM concentrations of different carbon sources (benzoate, choline, citrate, fructose, glucose, proline, and sucrose) (26).

TABLE 1.

Bacterial strains and plasmids used in this studya

Strain or plasmid Relevant characteristicsb Source or reference
Strains
    P. putida KT2440
        Wild type Prototrophic 27
        RpoS rpoS::miniTn5::lux 36
        cfaA mutant Mutant in cfaA inactivated by a Km cassette This study
        cfaB mutant Mutant in cfaB inactivated by a Gm cassette This study
    E. coli
        DH5α recA; host for cloning 18
        CC118λpir Rifr; host for pKNG101 plasmid and its derivatives 18
Plasmids
    pKNG101 Smr 20
    pUC18Not Apr; cloning vector 18
    pRK600 Cmr; helper plasmid 18
    pUK18 Apr Kmr; source of the Kmr cassette 25
    pMS265 Apr Gmr; source of the Gmr cassette 4
    pKNG101cfaAKm Smr Kmr This study
    pKNG101cfaBGm Smr Gmr This study
    pUcfaA Apr; pUC18Not derivative bearing cfaA This study
    pUcfaB Apr; pUC18Not derivative bearing cfaB This study
    pUcfaAKm Apr Kmr This study
    pUcfaBGm Apr Gmr This study
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a

All mutant strains have been deposited in the P. putida reference culture collection and can be obtained from E. Duque.

b

Rifr, rifampin resistance; Smr, streptomycin resistance; Apr, ampicillin resistance; Cmr, chloramphenicol resistance; Kmr, kanamycin resistance.

Lyophilization of P. putida KT2440.

P. putida KT2440 cells were grown until the desired turbidity, and then bacterial cells were harvested by centrifugation (7,500 × g for 10 min), washed twice with phosphate buffer, and resuspended in 30 ml of a lyoprotectant solution. Before the cells were freeze-dried the number of viable cells was counted in triplicate by spreading serial dilutions in LB solid medium. Different compounds were used as lyoprotectans, i.e., amino acids, disaccharides, monosaccharides, polyalcohols, and skimmed milk (Table 2). Sterile ampoules were filled with 500 μl of the bacterial suspension containing the lyoprotectant and covered with sterile cotton placed in the middle part of the ampoule. Ampoules with the bacterial suspension were first frozen at −20°C for 20 min and then submerged in liquid nitrogen. Subsequently, the freeze-drying process was carried out in a Virtis lyophilizer at 30 mtorr for 48 h. After the process, the ampoules were sealed under vacuum. At least three ampoules were used to determine the number of viable cells after lyophilization. To this end, 500 μl of M9 buffer solution was added to cells, and after 40 min, with occasional shaking, rehydratation was achieved. Bacterial cells were serially diluted and viable cells counted on LB plates.

TABLE 2.

Effect of different lyoprotectants on the survival of P. putida KT2440 upon lyophilization

Compound Concn (μM [or %]) Characteristic Mean BSR (SD)
Lactose 0.20 Disaccharide 80.35 (1.63)
Maltose 0.20 Disaccharide 83.73 (1.18)
Sucrose 0.20 Disaccharide 78.03 (2.16)
Trehalose 0.13 Disaccharide 83.72 (1.51)
Fructose 0.20 Monosaccharide 65.32 (5.79)
Galactose 0.20 Monosaccharide 69.80 (3.83)
Glucose 0.20 Monosaccharide 67.80 (2.11)
Manose 0.20 Monosaccharide 71.68 (1.70)
Manitol 0.20 Polyalcohol 42.34 (6.76)
Myoinositol 0.27 (5%) Polyalcohol 27.92 (4.77)
Sorbitol 0.27 Polyalcohol 61.69 (1.31)
Citrate 0.25 Tricarboxylic acid 41.82 (5.39)
Betaine 0.2 Amino acid 38.33 (6.20)
Glycine 0.2 Amino acid 23.23 (4.89)
Lysine 0.2 Amino acid 30.92 (1.08)
Proline 0.2 Amino acid 40.04 (4.77)
LB medium Complex mixture 56.04 (5.81)
Skimmed milk (20%) Complex mixture 67.01 (1.83)
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a

The BSR is the ratio (log CFU/ml after freeze-dry/log CFU/ml before freeze-dry) × 100. Each value represents the average of five independent determinations.

The bacterial survival ratio (BSR) is reported as the ratio of the log of the number of bacterial cells present in the suspension after lyophilization (AL) to the log number of viable cells before lyophilization (BL) multiplied by 100, i.e., BSR = (logAL/logBL) × 100.

Analysis of fatty acid profile.

P. putida KT2440 cells were grown for different periods of time and phospholipids were extracted by the method of Bligh and Dyer (5). To measure fatty acids, phospholipids were transesterified, and methyl ester derivatives were identified by mass spectrometry after gas chromatographic separation as described previously (35).

Membrane fluidity determinations.

Membrane fluidity was determined by measuring the fluorescence polarization of the probe DPH (1,6-diphenyl-1,3,5-hexatriene) inserted into the cytoplasmic membranes. Cells in two different growth phases were harvested by centrifugation (10,000 × g for 1 min), washed in sterile 15 mM Tris-HCl buffer (pH 7.0), and resuspended in same buffer to a turbidity at 660 nm of 0.2. Then, 1 μl of the fluorescent membrane probe (12 mM stock solution in tetrahydrofuran) was added to 3 ml of the resuspended culture in a quartz cuvette to obtain a final concentration of 4 μM and incubated with a magnetic stirring at 200 rpm for 10 min in the dark at 30°C to allow probe incorporation into the cytoplasmic membrane (43). Fluorescence polarization was measured by using a spectrofluorimeter (Photon Technology International, Inc.) equipped with a thermostated stabilized cell cuvette holder and a cuvette stirrer. The excitation wavelength for the DPH probe was 358 nm, and the emission wavelength was 428 nm. The slit width for the excitation and emission beams was 12 and 10 nm, respectively. The intensities parallel (IVV) and perpendicular (IVH) to the vertically polarized excitation beam were recorded and the degree of polarization (P) calculated from the intensity measurements with the equation P = (IVV − IVHG)/(IVV + IVHG), where G was the correlation factor for instrument polarization. G values were calculated as the ratio of vertical to horizontal measurements when the excitation light was polarized horizontally; the higher the value of polarization, the lower the membrane fluidity. Values were analyzed by using the FELIX (PTI) software program (43).

Construction of cfaA- and cfaB-null mutants of P. putida by gene replacement.

The original annotation of the P. putida KT2440 genome identified two potential open reading frames encoding C17:cyclopropane synthase (PP2734 and PP5365). These enzymes have been named CFA-A and CFA-B based on our work. Mutants of P. putida KT2440 with knockouts in the cfaA and cfaB genes were constructed by reverse genetic procedures. All plasmids for allelic replacements were based on the pKNG101 suicide vector and are listed in Table 1. Plasmids pKNG101cfaAKm and pKNG101cfaBGm bear truncated cfaA and cfaB alleles that were marked with kanamycin (Km) and gentamicin (Gm), respectively. Escherichia coli CC118λpir (pKNG101cfaAKm) was mated with P. putida KT2440 in the presence of the helper E. coli HB101(pRK600). Pseudomonas putida KT2440 transconjugants were selected on M9 minimal medium with benzoate as the carbon source and Km. The nature of the cointegration event was confirmed by PCR. A random clone was chosen and grown on LB without Sm for 20 generations. Then cells were spread on LB Km plates supplemented with 7% (wt/vol) of sucrose. Among the sucrose-resistant clones we searched for the Sm-sensitive ones as putative resolved clones. Several such clones were found, and upon confirmation of gene replacement by Southern blot a random clone was retained for further assays. This mutant strain was called KT2440-cfaA.

To construct KT2440 derivatives with a knockout in cfaB, we proceeded as described above except that we used pKNGcfaBGm to generate a chromosomal knockout in cfaB. A single mutant clone, KT2440-cfaB was retained for further work.

Statistical analysis.

Data for different experiments were compared by using the Student t test.

RESULTS

Disaccharides as lyoprotectants for P. putida KT2440.

To select lyoprotectants that conferred high survival to P. putida KT2440 cells after freeze-drying, we tested the disaccharides, monosaccharides, polyalcohols, amino acids, and complex mixtures shown in Table 2 at the indicated concentrations. In this series of assays P. putida KT2440 cultures were grown in LB medium until the mid-exponential phase, when the cell density was ca. 108 CFU/ml. Aliquots of 5 ml were harvested by centrifugation, and cells were suspended in 5 ml of the indicated lyoprotectant. Before and after lyophilization the number of viable cells was counted to estimate the BSR, as described in Materials and Methods. In general, we found that disaccharides were better lyoprotectants (BSR values of ca. 80) than monosaccharides (BSR values of ca. 68) and that these latter were better than polyalcohols, organic acids, LB, and amino acids. It should be noted that skimmed milk yielded a survival rate similar to that obtained with monosaccharides.

Effect of culture conditions and growth phase on the survival of P. putida KT2440 upon lyophilization.

For the following series of assays we used two different lyoprotectants: trehalose, which gave a high BSR under our experimental conditions, and myoinositol, a lyoprotectant often used to freeze-dry microorganisms (13, 29). First, cells were grown in rich LB medium overnight and then diluted 100-fold in fresh LB medium, and the number of viable cells was determined at the early and late exponential phases and at the stationary phase. At these times aliquots of cultures were withdrawn and freeze-dried, and the BSR was determined. We found that regardless of the growth phase, the BSR was higher with trehalose than with myoinositol and that BSR was higher in cells that had reached the stationary phase than in cells in the exponential phase (Table 3).

TABLE 3.

BSR of P. putida KT2440 at different growth phasesa

Lyoprotectant Mean BSR (SD) at:
Exponential (early) phase Exponential (late) phase Late stationary phase
Myoinositol 12.21 (8.00) 22.55 (7.5) 47.18 (2.00)
Trehalose 62.44 (5.0) 78.46 (5.0) 90.15 (1.50)
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a

P. putida KT2440 was grown on LB overnight. The culture was then diluted 100-fold. When it reached a turbidity at 660 nm of about 0.6 (early exponential phase), 1.2 (late exponential phase), or 4 (late stationary phase), aliquots were lyophilized with myoinositol or trehalose as the lyoprotectant.

We carried out assays similar to those described above but with cells grown in M9 minimal medium with different C sources, namely, betaine, benzoate, citrate, glucose, proline, and sucrose. We also found that, in general, survival was greater in cells that had reached the stationary phase than in cells in the exponential phase (not shown).

In our laboratory it has been a frequent practice to use solid LB or solid M9 minimal medium with citrate to grow cells for freeze-drying. We used 24-h-old plates that had been kept at 30°C and suspended cells for freeze-drying with trehalose or myoinositol. With trehalose the mean BSR value was 91 ± 3, and with myoinositol the mean value was 48 ± 1.

Regardless of how bacteria had been grown (liquid or solid medium) and whether cells were grown on rich or minimal medium, freeze-dried cells were stored under vacuum in the dark at 18 to 22°C. We found that survival decreased slightly with time and after 1 year the BSR of stored cells declined from 90 ± 2 to 80 ± 3.

Why do cells in the stationary phase survive better than cells in the exponential growth phase?

The results described above suggested that culture conditions and the physiological state of P. putida cells notably influenced tolerance to the freeze-drying process, a finding that may be related to changes in the properties of the membranes with growth. We tested membrane fluidity of P. putida by determining the polarization value (P) in cells that had been grown on citrate as a carbon source or LB and that had reached the exponential or the stationary phase. Polarization values were significantly higher in cells in the stationary phase (0.313 ± 0.007) than in the exponential phase (0.288 ± 0.010), indicating that the membranes of P. putida were more rigid in the stationary phase than in the exponential growth phase. Membrane fluidity can be influenced by the composition of phospholipids. We reported before that the fatty acid composition of P. putida varied according to the growth phase. Ramos et al. (35) found that cells grown in LB medium to the exponential phase exhibited low levels of C17:cyclopropane (the most abundant cyclopropane fatty acid [CFA] found in P. putida), whereas CFA levels were high in the stationary phase. To test this potential correlation, we determined the fatty acid profile in cells growing on LB and M9 minimal media with citrate. In agreement with previous observations, C17:cyclopropane levels were higher in cells that had reached the stationary phase in M9 with citrate or LB. Concomitantly with the increase in C17:cyclopropane, we noted an increase in BSR (Table 4).

TABLE 4.

Fatty acid profiles from P. putida KT2440 cells grown in different media and at different growth stages

Lipid Mean % ± SDa
Exponential phase
Stationary phase
Citrate LB Citrate LB
C14:0 2.65 ± 0.28 1.77 ± 0.20 1.38 ± 0.11 2.09 ± 0.43
C15:0 ND 0.06 ± 0.05 ND 0.81 ± 0.02
C16:1,9cis 10.48 ± 1.31 23.74 ± 1.93 0.42 ± 0.19 3.09 ± 0.53
C16:1,9trans 4.66 ± 0.30 6.47 ± 0.56 1.81 ± 0.8 1.24 ± 0.43
C16:0 31.53 ± 0.68 35.45 ± 2.79 42.61 ± 0.91 33.22 ± 0.27
C17:cyclopropane 0.71 ± 0.16 4.96 ± 0.64 30.05 ± 0.46 33.21 ± 1.19
C17:0 0.44 ± 0.04 0.31 ± 0.09 0.70 ± 0.49 0.75 ± 0.03
C18:1,11cis 11.75 ± 1.77 14.55 ± 0.83 6.30 ± 0.58 14.11 ± 0.74
C18:1,11trans 0.91 ± 0.15 0.81 ± 0.06 ND 1.04 ± 0.26
C18:0 36.84 ± 3.28 11.88 ± 6.13 10.24 ± 1.26 6.89 ± 0.40
C19:cyclopropane ND ND 6.50 ± 0.87 4.15 ± 0.82
BSR 66.67 ± 2.88 79.23 ± 0.98 92.07 ± 2.20 91.28 ± 1.50
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a

Each value represents the average of three repetitions from independent experiments. BSR values are shown at the bottom of the table. ND, not detected.

Survival of mutants deficient in the synthesis of C17:cyclopropane in P. putida KT2440 upon freeze-drying.

To gain further insight into the potential role of CFA in freeze-drying we generated mutants in the synthesis of C17:cyclopropane. In gram-negative bacteria C17:cyclopropane is made from C16:1cis in a reaction catalyzed by CFA synthases (9). The genome of KT2440 had been sequenced previously and two potential CFA synthase genes were annotated (27). These genes are called cfaA and cfaB here, and their translated products corresponded to PP2734 and PP5365, respectively. We inactivated each of the genes by using cfaA:Km and cfaB:Gm mutant alleles and used them to generate each of the single mutants by allelic exchange.

We analyzed C17:cyclopropane levels in the mutant strains growing on citrate when cells had reached the exponential and the stationary phase. In the mutant strains C17:cyclopropane in the exponential phase was negligible, whereas in the stationary phase the level of C17:cyclopropane in the cfaA mutant was 20% of the total fatty acids versus ca. 30% in the wild type. Surprisingly, in the cfaB mutant C17:cyclopropane level in the stationary phase was less than 0.2% in LB, and it was not detected in cells grown on citrate as the sole carbon source (Table 5). We measured polarization values with the P. putida CFA-deficient strain and found that P values in the exponential and stationary phase were similar to those observed for the wild type. In this mutant strain the increase in rigidity observed could be due to a fivefold increase in the levels of C16:1trans (not shown). We calculated the BSR of each strain with a mutation in the cfa genes, in cells in the exponential and stationary phase. For the cfaB mutant, regardless of the growth phase, BSR values were much lower than those of the parental strain. For the cfaA mutant in the exponential phase, the BSR values were similar to those of the wild-type strain; however, when cells reached the stationary phase the values were slightly lower (Table 5). It then follows that BSR values correlate well with C17:cyclopropane levels.

TABLE 5.

BSR and levels of C17:cyclopropane in P. putida KT2440 and several isogenic mutant strains

P. putida strain Mean % (SD)a
Level of C17:cyclopropane at:
BSR at:
Exponential phase Stationary phase Exponential phase Stationary phase
KT2440 0.5 (0.2) 30.7 (1.5) 68.5 (2.21) 91.83 (1.63)
cfaA mutant 0.1 (0.1) 20.2 (3.60) 64.94 (6.3) 85.31 (1.23)
cfaB mutant ND ND 58.94 (5.3) 70.55 (1.60)
ropS (miniTn5::lux) mutant 0.1 (0.1) 7.0 (1.0) 66.48 (2.7) 73.39 (1.99)
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a

Each value represents the average of three repetitions from independent experiments. Cells were grown in M9 minimal medium with 20 mM citrate. The turbidity of the cultures in the exponential phase was around 0.3 ± 0.05 at 660 nm, whereas in the stationary phase the turbidity at 660 nm was 2.5 ± 0.2. ND, not detected.

In E. coli cfa transcription is mediated by the rpoS sigma factor when cells reach the stationary phase (11, 45). This also seems to be the case in KT2440 (P. Bernal et al., unpublished). We tested CFA levels in an rpoS KT2440 mutant background and found that the rpoS mutant produced very small amounts of C17:cyclopropane in the stationary phase and that its BSR values were concomitantly lower than those of the wild-type strain.

DISCUSSION

Freeze-drying is the method often used for the preservation and long-term storage of bacterial cells (29); however, it has negative effects on the viability of many cell types (24). It is known that lyophilization of bacteria results in the loss of viability during the process (32) and that such loss is influenced by the lyoprotectant used (12, 22, 44), the initial cell density (8, 28), and the physiological state of the cells (6, 13). Several compounds and mixtures were used as lyoprotectants for P. putida KT2440 in the present study. We found that viability was affected to a certain extent under all conditions compared here but that disaccharides, e.g., trehalose, maltose, and lactose, were the best lyoprotectants. This finding is in agreement with observations reported in experiments with other microorganisms (Escherichia coli, Bacillus thuringiensis, and Pseudomonas chlororaphis) that were protected during lyophilization by disaccharides (22, 23, 28). The molecular mechanism behind the protective effect of disaccharides during freeze-drying is unknown, although it has been suggested that these chemicals protect membrane integrity by replacing water molecules in the membrane during the freeze-drying process (23).

With disaccharides as lyoprotectants for P. putida KT2440, we observed that survival was notably influenced by the physiological state of bacteria. In particular, cells in the stationary phase survived better than those in the exponential phase. In the stationary phase bacteria accumulate compatible solutes (21, 31, 40) and produced stress proteins under carbon starvation conditions (17, 28). We cannot rule out these possibilities of protecting the cells against damage during lyophilization; nevertheless, we suggest that membrane alterations are the main factor responsible for the increase in BSR. We found that the increase in BSR in the stationary phase correlated with an increase in C17:cyclopropane levels. The presence of high levels of C17:cyclopropane in the stationary phase are in agreement with a recent report by Härtig et al. (16), although we have noticed higher levels of C18:0 in the same strain under the growth conditions we used. Cyclopropane fatty acids seem to stabilize membrane lipids against turnover and degradation (23). Several reports have indicated that CFAs are involved in stress tolerance.

Zhao et al. (47) reported that overexpression of a cloned cyclopropane fatty acid synthase in Clostridium sp. influenced butanol resistance and in E. coli and Streptococcus sp. CFAs were said to be involved in acid stress since E. coli cfa mutants were shown to be more sensitive to stress (7, 33). In E. coli CFA deficiency also led to increased sensitivity to recurring freeze-thaw cycles (42). Azotobacter vinelandii synthesizes C17:cyclopropane fatty acid during differentiation into cysts as a mechanism of resistance to water and other environmental stresses (41). It seems then that under certain conditions CFA can function as a resistance barrier against environmental stresses. We reasoned that if C17:cyclopropane fatty acid was involved in increased tolerance to lyophilization in P. putida, mutants deficient in the synthesis of C17:cyclopropane fatty acids should be less tolerant to freeze-drying than wild-type cells. First, we showed that single mutants in cfaA or cfaB grew as fast as the wild type in LB rich medium. We then tested the mutants' response to lyophilization. Our finding that the single cfaB mutant, which produced very low levels of C17:cyclopropane, was hypersensitive to lyophilization even when it reached the stationary phase of growth on LB medium, supports a role of this fatty acid in tolerance to lyophilization. The cfaA single mutant that synthesized ca. 66% of the total C17:cyclopropane fatty acid produced by the wild type was more sensitive than the parental strain to lyophilization but much more resistant than the cfaB mutant. The increased resistance to freeze-drying offered by C17:cyclopropane fatty acid can be interpreted as evidence for its role in the alteration of the membrane's chemical properties. This is in consonance with the increased resistance of CFA to ozonolysis, singlet oxygen, and mild oxidative treatments (14, 15).

In P. putida CFA synthesis occurred in the stationary phase, and expression of cfa genes seems to be mediated by the RpoS sigma factor. This was suggested by the finding that an rpoS knockout mutant produced very low levels of CFA. In consonance with the low levels of C17:cyclopropane fatty acids, the RpoS mutant was also hypersensitive to lyophilization. In E. coli the expression of the cfa genes is influenced not only by RpoS but also by the Crp and Fur proteins (46). Under our growth conditions a role for iron in CFA synthesis can be ruled out, since KT2440 was grown in iron-rich medium, and no iron siderophores were detected in the medium. Catabolite repression might play a role in cfa gene expression, but in P. putida the intimate mechanisms of catabolite repression have not been elucidated (1, 10). In the near future we plan to perform detailed transcriptional studies of cfa synthesis in different genetic backgrounds.

In short, our results indicate that cyclopropane fatty acids are important in P. putida KT2440 for survival in freeze-drying processes and that the highest survival rates in this strain are achieved with disaccharides as lyoprotectants.

Acknowledgments

This study was supported by a grant from the CICYT (BIO-2003-00515) and a grant from the European Commission (BIOCARTE QLK3-CT-2002-1923). J.M.-R. was the recipient of a postdoctoral fellowship of the “Cátedra Volante” program from Banco de Santander-Central-Hispano/CSIC. P.B. is the recipient of an I3P fellowship.

We thank Carmen Lorente for secretarial assistance and Karen Shashok for improving the English in the manuscript.

REFERENCES

  • 1.Aranda-Olmedo, I., J. L. Ramos, and S. Marqués. 2005. Integration of signals through the PTS and CRC systems in catabolite repression of the Pseudomonas putida TOL plasmid pWW0. Appl. Environ. Microbiol. 71:4191-4198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Arias-Barrau, E., E. R. Olivera, J. M. Luengo, C. Fernández, B. Galan, J. L. García, E. Díaz, and B. Miñambres. 2004. The homogentisate pathway: a central catabolic pathway involved in the degradation of l-phenylalanine, l-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida. J. Bacteriol. 186:5062-5077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bagdasarian, M. M., and K. N. Timmis. 1982. Host: vector systems for gene cloning in Pseudomonas. Curr. Top. Microbiol. Immunol. 96:47-67. [DOI] [PubMed] [Google Scholar]
  • 4.Becker, A., M. Schmidt, W. Jager, and A. Pühler. 1995. New gentamicin-resistance and lacZ promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions. Gene 162:37-39. [DOI] [PubMed] [Google Scholar]
  • 5.Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. [DOI] [PubMed] [Google Scholar]
  • 6.Broadbent, J. R., and C. Lin. 1999. Effect of heat shock or cold shock treatment on the resistance of Lactococcus lactis to freezing and lyophilization. Cryobiology 39:88-102. [DOI] [PubMed] [Google Scholar]
  • 7.Chang, Y. Y., and J. E. Cronan, Jr. 1999. Membrane cyclopropane fatty acid content is a major factor in acid resistance of Escherichia coli. Mol. Microbiol. 33:249-259. [DOI] [PubMed] [Google Scholar]
  • 8.Costa, E., J. Usall, N. Teixido, N. García, and I. Viñas. 2000. Effect of protective agents, rehydration media and initial cell concentration on viability of Pantoea agglomerans strain CPA-2 subjected to freeze-drying. J. Appl. Microbiol. 89:793-800. [DOI] [PubMed] [Google Scholar]
  • 9.Cronan, Jr., J. E., R. Reed, F. R. Taylor, and M. B. Jackson. 1979. Properties and biosynthesis of cyclopropane fatty acids in Escherichia coli. J. Bacteriol. 138:118-121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Dinamarca, M. A., A. Ruíz-Manzano, and F. Rojo. 2002. Inactivation of cytochrome o ubiquinol oxidase relieves catabolic repression of the Pseudomonas putida GPo1 alkane degradation pathway. J. Bacteriol. 184:3785-3793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Eichel, J., Y.-Y. Chang, D. Riesenberg, and J. E. Cronan, Jr. 1999. Effect of ppGpp on Escherichia coli cyclopropane fatty acid synthesis is mediated through the RpoS sigma factor (σS). J. Bacteriol. 181:572-576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Font de Valdéz, G., G. Savoy de Giori, A. Pesce de Ruiz Holgado, and G. Oliver. 1983. Comparative study of the efficiency of some additives in protecting lactic acid bacteria against freeze-drying. Cryobiology 20:560-566. [DOI] [PubMed] [Google Scholar]
  • 13.García-López, M. D., and F. Uruburu-Fernández. 2000. La conservación de cepas microbianas. Actualidad SEM 30:12-16. [Google Scholar]
  • 14.Grogan, D. W., and J. E. Cronan, Jr. 1986. Characterization of Escherichia coli mutants completely defective in the synthesis of cyclopropane fatty acids. J. Bacteriol. 166:872-877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Grogan, D. W., and J. E. Cronan, Jr. 1997. Cyclopropane ring formation in membrane lipids of bacteria. Microbiol. Mol. Biol. Rev. 61:429-441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Härtig, C., N. Loffhagen, and H. Harms. 2005. Formation of trans fatty acids is not involved in growth-linked membrane adaptation of Pseudomonas putida. Appl. Environ. Microbiol. 71:1915-1922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Hecker, M., and U. Volker. 2001. General stress response of Bacillus subtilis and other bacteria. Adv. Microb. Physiol. 44:35-91. [DOI] [PubMed] [Google Scholar]
  • 18.Herrero, M., V. de Lorenzo, and K. N. Timmis. 1990. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J. Bacteriol. 172:6557-6567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Jiménez, J. I., B. Miñambres, J. L. García, and E. Díaz. 2002. Genomic analysis of the aromatic catabolic pathways for Pseudomonas putida KT2440. Environ. Microbiol. 4:824-841. [DOI] [PubMed] [Google Scholar]
  • 20.Kaniga, K., I. Delor, and G. R. Cornelis. 1991. A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene 109:137-141. [DOI] [PubMed] [Google Scholar]
  • 21.Kets, P. W., A. Galinski, M. de Wit, J. A. M. de Bont, and H. J. Heipieper. 1996. Mannitol as a novel bacterial compatible solute in Pseudomonas putida S12. J. Bacteriol. 178:6665-6670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Leslie, S. B., E. Israeli, B. Lighthart, J. H. Crowe, and L. M. Crowe. 1995. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl. Environ. Microbiol. 61:3592-3597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.MacDonald, P. M., B. D. Sykes, and R. N. McElhaney. 1985. Florine-19 nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. A direct comparison of the effects of cis and trans cyclopropane ring and double-bond substituents on orientational order. Biochemistry 24:4651-4659. [DOI] [PubMed] [Google Scholar]
  • 24.MacKenzie, A. P. 1976. Comparative studies on the freeze-drying survival of various bacteria: Gram type, suspending media and freeze rate. Dev. Biol. Stand. 36:263-277. [PubMed] [Google Scholar]
  • 25.Ménard, R., P. J. Sansonetti, and C. Parsot. 1993. Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cell. J. Bacteriol. 175:5899-5906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Molina, L., C. Ramos, E. Duque, M. C. Ronchel, J. M. García, L. Wyke, and J. L. Ramos. 2000. Survival of Pseudomonas putida KT2440 in soil and in the rhizosphere of plants under greenhouse and environmental conditions. Soil Biol. Biochem. 32:315-321. [Google Scholar]
  • 27.Nelson, K. E., C. Weinel, I. T. Paulsen, R. J. Dodson, H. Hilbert, V. A. Martins dos Santos, D. E. Fouts, S. R. Gill, M. Pop, M. Holmes, L. Brinkac, M. Beanan, R. T. DeBoy, S. Daugherty, J. Kolonay, R. Madupu, W. Nelson, O. White, J. Peterson, H. Khouri, I. Hance, L. P. Chris, E. Holtzapple, D. Scanlan, K. Tran, A. Moazzez, T. Utterback, M. Rizzo, K. Lee, D. Kosack, D. Moestl, H.Wedler, J. Lauber, D. Stjepandic, J. Hoheisel, M. Straetz, S. Heim, C. Kiewitz, J. A. Eisen, K. N. Timmis, A. Dusterhoft, B. Tummler, and C. M. Fraser. 2002. Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ. Microbiol. 4:799-808. [DOI] [PubMed] [Google Scholar]
  • 28.Palmfeldt, J., P. Radstrom, and B. Hahn-Hagerdal. 2003. Optimisation of initial cell concentration enhances freeze-drying tolerance of Pseudomonas chlororaphis. Cryobiology 47:21-29. [DOI] [PubMed] [Google Scholar]
  • 29.Perry, S. F. 1995. Free-drying and cryopreservation of bacteria. Methods Mol. Biol. 38:21-30. [DOI] [PubMed] [Google Scholar]
  • 30.Pieper, D. H., V. Martins dos Santos, and P. N. Golyshin. 2004. Genomic and mechanistic insights into the biodegradation of organic pollutants. Curr. Opin. Biotechnol. 15:215-224. [DOI] [PubMed] [Google Scholar]
  • 31.Poolman, B., and E. Glaasker. 1998. Regulation of compatible solute accumulation in bacteria. Mol. Microbiol. 29:397-407. [DOI] [PubMed] [Google Scholar]
  • 32.Potts, M. 1994. Desiccation tolerance of prokaryotes. Microbiol. Rev. 58:755-805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Quivey, R. G., Jr., R. Faustoferri, K. Monahan, and R. Marquis. 2000. Shifts in membrane fatty acid profiles associated with acid adaptation of Streptococcus mutants. FEMS Microbiol. Lett. 189:89-92. [DOI] [PubMed] [Google Scholar]
  • 34.Ramos, J. L., E. Díaz, D. Dowling, V. de Lorenzo, S. Molin, F. O'Gara, C. Ramos, and K. N. Timmis. 1994. The behavior of bacteria designed for biodegradation. Biotechnology 12:1349-1356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Ramos, J. L., E. Duque, J. J. Rodríguez-Herva, P. Godoy, A. Haidour, F. Reyes, and A. Fernández-Barrero. 1997. Mechanisms for solvent tolerance in bacteria. J. Biol. Chem. 272:3887-3890. [DOI] [PubMed] [Google Scholar]
  • 36.Ramos-González, M. I., and S. Molin. 1998. Cloning, sequencing, and phenotypic characterization of the rpoS gene from Pseudomonas putida KT2440. J. Bacteriol. 180:3421-3431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Redway, K. F., and S. P. Lapage. 1974. Effect of carbohydrates and related compounds on the long-term preservation of freeze-dried bacteria. Cryobiology 11:73-79. [DOI] [PubMed] [Google Scholar]
  • 38.Regenhardt, D., H. Heuer, S. Heim, D. U. Fernandez, C. Strompl, E. R. Moore, and K. N. Timmis. 2002. Pedigree and taxonomic credentials of Pseudomonas putida strain KT2440. Environ. Microbiol. 4:912-915. [DOI] [PubMed] [Google Scholar]
  • 39.Revelles, O., M. Espinosa-Urgel, S. Molin, and J. L. Ramos. 2004. The davDT operon of Pseudomonas putida, involved in lysine catabolism, is induced in response to the pathway intermediate delta-aminovaleric acid. J. Bacteriol. 186:3439-3446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Roeßler, M., and V. Müller. 2001. Osmoadaptation in bacteria and archeae: common principles and differences. Environ. Microbiol. 3:743-754. [DOI] [PubMed] [Google Scholar]
  • 41.Su, C. J., R. Reusch, and H. L. Sadoff. 1979. Fatty acids in phospholipids of cells, cysts and germinating cysts of Azotobacter vinelandii. J. Bacteriol. 137:1434-1436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Taylor, F., and J. E. Cronan, Jr. 1976. Selection and properties of Escherichia coli mutants defective in the synthesis of cyclopropane fatty acids. J. Bacteriol. 125:518-523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Trevors, J. T. 2003. Fluorescent probes for bacterial cytoplasmic membrane research. J. Biochem. Biophys. Methods 57:87-103. [DOI] [PubMed] [Google Scholar]
  • 44.Tsvetkov, T., and R. Brankova. 1983. Viability of micrococci and lactobacilli upon freezing and freeze drying in the presence of different cryoprotectants. Cryobiology 20:318-323. [DOI] [PubMed] [Google Scholar]
  • 45.Wang, A.-Y., and J. E. Cronan, Jr. 1994. The growth phase-dependent synthesis of cyclopropane fatty acids in Escherichia coli is the result of an RpoS (KatF)-dependent promoter plus enzyme instability. Mol. Microbiol. 11:1009-1017. [DOI] [PubMed] [Google Scholar]
  • 46.Zhang, Z., G. Gosset, R. Barabote, C. S. González, W. A. Cuevas, and U. Sauer. 2005. Functional interactions between the carbon and iron utilization regulators, Crp and Fur, in Escherichia coli. J. Bacteriol. 187:980-990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Zhao, Y., L. A. Hindorff, A. Chuang, M. Monroe-Augustus, M. Lyristis, M. L. Harrison, F. B. Rudolph, and G. N. Bennet. 2003. Expression of a cloned cyclopropane fatty acid synthase gene reduces solvent formation in Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 69:2831-2841. [DOI] [PMC free article] [PubMed] [Google Scholar]

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