Department of Biological Sciences, University of Alberta, Edmonton, Alberta, CanadaT6G 2E91
Author for correspondence: William J. Page. Tel: +1 780 492 4782. Fax: +1 780 492 2216. e-mail: bill.page{at}ualberta.ca
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ABSTRACT |
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Keywords: Azotobacter vinelandii, alginate, algU, poly-ß-hydroxybutyrate, RpoS
Abbreviations: PHA, polyhydroxyalkanoate; PHB, poly-ß-hydroxybutyrate
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INTRODUCTION |
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The PHB-hyperproduction phenotype was originally created in A. vinelandii strain 113 by nitrosoguanidine mutagenesis (Page & Knosp, 1989 ). However, strain 113 is unsuited to commercial PHB production because it forms copious amounts of extracellular alginate. Thus strain UWD was created by transformation of the PHB-hyperproduction trait of strain 113 into A. vinelandii strain UW (Page & Knosp, 1989
), an alginate-minus host (Bush & Wilson, 1959
). The capsule-minus genotype of strain UW is extremely stable and spontaneous revertants to the capsule-positive phenotype have never been reported.
However, the alginate biosynthetic pathway is intact in strain UW and very limited alginate production has been observed after prolonged incubation (Page, 1983 ). The A. vinelandii biosynthetic pathway has been shown previously to be identical to that found in Pseudomonas aeruginosa and many of the genes are conserved (Fialho et al., 1990
; Gacesa, 1998
; Rehm et al., 1996
). The transcriptional activation of the GDP-mannose dehydrogenase gene (algD) is a key point in pathway regulation (Campos et al., 1996
) and is mediated by the alternative sigma factor AlgU, a homologue of RpoE (Martinez-Salazar et al., 1996
). Recently it was shown that A. vinelandii strain UW136 has a natural insertion element in the algU gene (Martinez-Salazar et al., 1996
), which may explain the alginate-minus phenotype of strains derived from UW.
We have recently been using O2-limited culture for the production of higher yields of PHA in strain UWD (Chen & Page, 1997 ). The promotion of PHA formation by O2 limitation is commonly used with other bacteria (Anderson & Dawes, 1990
) and does increase PHA yield in strain UWD. However, we have observed that the culture can become viscous, leading to foam stabilization and difficulty harvesting the cells (unpublished results). In this study, we show that strain UWD can form a significant amount of alginate in an AlgU-independent manner during the turnover of PHB.
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METHODS |
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The strains were routinely grown in Burks buffer salts (Page & Sadoff, 1976 ), supplemented with ferric citrate to bring the total iron concentration to 50 µM, 2% (w/v) glucose and 15 mM ammonium acetate (Burks medium). The medium for optimal PHB production was Burks buffer salts, 50 µM ferric citrate, 3% (w/v) glucose and 0·1% (w/v) fish peptone (Page, 1992
). Cultures were incubated at high or low aeration (50 ml or 100 ml per 500 ml Erlenmeyer flask, respectively) with shaking at 225 r.p.m. at 2830 °C. Growth curves were constructed from multiple flasks that were inoculated at the same time so that all the samples at a time point were removed from a single flask. Low-aeration conditions were also created for the incubation of Petri plates using a microaerophilic gas-generating kit (Oxoid CampyPak) in various sized jars to give a calculated 6, 11 and 16% (v/v) O2.
Strain UWD cells for shift-down growth experiments were grown for 48 h in 100 ml Burks medium, then harvested by centrifugation, washed in Burks buffer and resuspended in an equal volume of Burks buffer. The cell suspension was dispensed 20 ml per 50 ml Erlenmeyer flask and incubated at 30 °C with shaking at 225 r.p.m. for up to 8 d.
Analysis of cells and culture fluids.
Capsule was solubilized by the addition of 1 ml 5·0 M NaCl and 2 ml 0·05 M disodium EDTA, pH 7·0, to 50 ml culture prior to centrifugation at 10000 g (10 min) to remove the cells. Alginate in the culture supernatant fluid was precipitated with 3 vols ice-cold 95% (w/v) ethanol overnight at 4 °C. The precipitated alginate was collected on a Whatman GF/A glass fibre filter and dissolved in 0·1 M NaOH at room temperature prior to assay by the carbazole assay (Knutson & Jeanes, 1968 ) using commercial alginate (Sigma) as a standard. PHB dry weight was determined after digestion of cell material in commercial bleach (Law & Slepecky, 1961
). Cell protein, glucose and ammonia remaining in the culture fluid were determined by colorimetric assays [the Lowry method, Trinder Sigma Diagnostics, and Bergersen (1980)
, respectively]. Alkyl resorcinols were extracted from cell material with acetone overnight at room temperature (Kozubek & Tyman, 1995
), then assayed by a colorimetric assay using Fast Blue B with orcinol as the standard (Tluscik et al., 1981
). ß-Galactosidase specific activity (Miller, 1972
) was measured as A420 (mg cell protein)-1 h-1, rather than optical density, because culture optical density was greatly affected by the PHB content of the cells. NADH oxidase activity was assayed as described previously (Page, 1991
).
DNA isolation and PCR amplification of algU.
Chromosomal DNA was extracted from A. vinelandii (Robson et al., 1984 ). The region of algU expected to contain a natural insertion element (Martinez-Salazar et al., 1996
) was amplified by PCR. The Expand Long Template PCR System (Boehringer Mannheim) was used in buffer system 3 with 200 µM dNTPs, 1·25 µM of each primer, 1·5 mM MgCl2, 400 ng chromosomal DNA, 5% (v/v) DMSO and 2·5 units of the DNA polymerase mix in a final volume of 100 µl. The primers for algU amplification were WJP13 (5'-ATGTTAAACCAGGAGCAAGATCA-3') and WJP46 (5'-CATAAAACTCGGCATCACCTGCA-3'). The PCR program (Minicycler, MJ Research) was: 5 min denaturation at 95 °C, followed by 30 cycles of 30 s denaturation at 95 °C, 1 min annealing at 37 °C, and 2 min extension at 68 °C.
Southern hybridization followed standard procedures (Southern, 1975 ), using 32P-end-labelled WJP12 (5'-GGCTATGTCTGAGTTAGCTGTT-3') as a probe for the insertion element and WJP51 (5'-GAAAATGCTGATACGCATTGCGTTC-3') as a probe for algD.
RNA extraction and primer extension analysis.
A. vinelandii cultures were grown for 24 and 48 h in Burks medium prior to RNA extraction and primer extension analysis as described previously (Tindale et al., 2000 ). The oligonucleotide WJP51 was used as the primer.
The sequence ladder used to identify the starts of algD transcription was derived from the strain UWD algD promoter region, which had been amplified by PCR using primers WJP50 and WJP49. Primer WJP50 hybridized with a sequence 389 nt upstream of the start of the A. vinelandii ATCC 9046 algD gene (Campos et al., 1996 ) and had a PstI site (lower case) tagged on for subsequent cloning of the PCR product: 5'-ctctctgcagATTCACCTATTCCACACTGG-3'. The reverse primer WJP49 hybridized with a sequence 199 nt into the algD gene and had an EcoRI site (lower case) tagged on: 5'-tctcgaattcGGGGATTTGCCCTGATTGAT-3'. The amplified strain UWD algD promoter region was found to have a nucleotide sequence identical to the algD promoter region of ATCC 9046 (GenBank U11240).
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RESULTS AND DISCUSSION |
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When alginate production by strain UWD was examined on a finer timescale, two rates of alginate formation occurred during the first 50 h of incubation (Fig. 2). During exponential growth (at times prior to 30 h, Fig. 1a
), the alginate production rate was 1·2 µg (mg cell protein)-1 h-1 (r2=0·969). During stationary phase, the rate increased to 4·8 µg (mg cell protein)-1 h-1 (r2=0·997). This rate was similar in magnitude to the long-term rate of 3·8 µg (mg cell protein)-1 h-1 observed in Fig. 1(b)
. In contrast, strain UW formed alginate at a low but constant rate of 0·6 µg (mg cell protein)-1 h-1 (r2=0·951) (Fig. 2
). Strain O formed alginate at a much higher rate of 168 µg (mg cell protein)-1 h-1 (r2=0·887) until stationary phase, when the rate decreased to
3 µg (mg cell protein)-1 h-1 (r2=0·031), presumably due to O2 limitation (data not shown).
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PHB-filled cells form alginate
In order to confirm that alginate formed by strain UWD was derived from PHB turnover, cells that had been grown for 48 h in Burks medium were used to inoculate Burks buffer (without an exogenous carbon source) and incubated for up to 8 d. During the course of this nutrient shift-down, extracellular alginate rapidly increased while PHB decreased from 13·4 to 9·8 mg (mg cell protein)-1 (Fig. 3b). Alkyl resorcinols also formed coincident with PHB turnover in the first 5 d of incubation (Fig. 3a
). The rate of PHB degradation was 407 µg (mg cell protein)-1 d-1 (r2=0·740) and the rate of alginate formation was 147 µg (mg cell protein)-1 d-1 (r2=0·854).
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Inactivation of alginate formation in strain UWD
DNA from A. vinelandii strain U5 (algD::Tn5lacZ) was transformed into strain UWD and KanR transformants were selected. These transformants (designated DU5) formed 5 µg alginate (mg cell protein)-1 under O2-limited growth conditions, suggesting that homologous recombination and inactivation of algD had occurred. This was confirmed by Southern hybridization of NotI-digested chromosomal DNA using WJP51 as a probe for algD. Strains UWD, U5 and DU5 produced a single restriction fragment that contained algD. The U5 and DU5 fragment was larger than that found in UWD, owing to the insertion of about 4 kb of Tn5lacZ DNA (de Lorenzo et al., 1990
) into algD (data not shown).
Under optimal PHB production conditions, strain DU5 produced 8·0±0·2 mg PHB (mg cell protein)-1 like strain UWD, which was four times that formed in strain UW. Strains DU5 and UWD formed PHB from glucose at maximum theoretical efficiency (0·33±0·3 g g-1) (Collins, 1987 ) since alginate formation occurred after PHB accumulation (as in Fig. 1b
). Upon glucose limitation, strain DU5 turned-over PHB and formed alkyl resorcinols, but as noted above alginate was not formed.
Transcription of algD was examined in strains U5 and DU5 to determine if there was an obvious increase in algD transcription at the times when alginate formation was active (Fig. 4). The rates of algD transcriptional activity were not significantly different in strains DU5 and U5 during the exponential phase of growth. This activity reached a plateau in strain U5 as the cells entered the stationary phase, but continued unabated in strain DU5 (Fig. 4
). The amount of LacZ activity in these cells was very low, suggesting that algD transcription was initiated from a weak promoter.
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Growth-phase-dependent alginate formation
Since strains UW and UWD are identical in terms of having an inactive algU, the difference in alginate formation may be a consequence of the inactive respiratory NADH oxidase in strain UWD (Page & Knosp, 1989 ). To test this, strain UWD was backcrossed with strain UW DNA and less opaque (clear) colonies were picked. These colonies were not observed in strain UWD culture, but occurred at a frequency of 3x10-2 in transformed culture. Ten transformants (designated DC) were analysed and found to have increased NADH oxidase activity compared to strain UWD, lower PHB yields and had minimal alginate production like strain UW (Table 1
). However, strain DC was rifampicin-resistant like strain UWD, which distinguished the transformants from the donor strain UW, which was rifampicin-sensitive (Page & Knosp, 1989
).
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NOTE ADDED IN PROOF |
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ACKNOWLEDGEMENTS |
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Received 10 March 2000;
revised 9 October 2000;
accepted 10 October 2000.