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Applied and Environmental Microbiology, February 2006, p. 1410-1419, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1410-1419.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Engineering the Genotype of Acinetobacter sp. Strain ADP1 To Enhance Biosynthesis of Cyanophycin

Yasser Elbahloul and Alexander Steinbüchel*

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Münster, Germany

Received 22 September 2005/ Accepted 12 December 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To study the importance of arginine provision and phosphate limitation for synthesis and accumulation of cyanophycin (CGP) in Acinetobacter sp. strain ADP1, genes encoding the putative arginine regulatory protein (argR) and the arginine succinyltransferase (astA) were inactivated, and the effects of these mutations on CGP synthesis were analyzed. The inactivation of these genes resulted in a 3.5- or 7-fold increase in CGP content, respectively, when the cells were grown on glutamate. Knockout mutations in both genes led to a better understanding of the effect of the addition of other substrates to arginine on CGP synthesis during growth of the cells of Acinetobacter sp. strain ADP1. Overexpression of ArgF (ornithine carbamoyltransferase), CarA-CarB (small and large subunits of carbamoylphosphate synthetase), and PepC (phosphoenolpyruvate carboxylase) triggered synthesis of CGP if amino acids were used as a carbon source whereas it was not triggered by gluconate or other sugars. Cells of Acinetobacter sp. strain ADP1, which is largely lacking genes for carbohydrate metabolism, showed a significant increase in CGP contents when grown on mineral medium supplemented with glutamate, aspartate, or arginine. The Acinetobacter sp. {Delta}astA(pYargF) strain is unable to utilize arginine but synthesizes more arginine, resulting in CGP contents as high as 30% and 25% of cell dry matter when grown on protamylasse or Luria-Bertani medium, respectively. This recombinant strain overcame the bottleneck of the costly arginine provision where it produces about 75% of the CGP obtained from the parent cells grown on mineral medium containing pure arginine as the sole source of carbon. Phosphate starvation is the only known trigger for CGP synthesis in this bacterium, which possesses the PhoB/PhoR phosphate regulon system. Overexpression of phoB caused an 8.6-fold increase in CGP content in comparison to the parent strain at a nonlimiting phosphate concentration.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cyanophycin (cyanophycin granule polypeptide [CGP]) was identified more than 100 years ago (47). CGP is a branched non-ribosomally synthesized copolymer with aspartic acid in the backbone linked by its ß-carboxyl group to the {alpha}-amino group of arginine by an amide bond (62). The key enzyme of CGP synthesis is cyanophycin synthetase (CphA), which catalyzes the ATP-dependent polymerization of aspartic acid and arginine. Genes encoding cyanophycin synthetase (cphA) were identified in many cyanobacteria such as Synechococcus sp. strain MA19, Anabaena variabilis ATCC 29413, Anabaena sp. strain PCC7120, Synechocystis sp. strains PCC6803 and PCC6308, and Synechococcus elongates (3, 11, 24, 48, 79). CGP was described as a unique polymer of cyanobacteria (8, 35, 60) and was until recently not found in other groups of bacteria or in archaea (58). CGP seems to be present in the vegetative cells of most genera of cyanobacteria (60, 65) and is absent in only a few species (6, 7, 8, 16, 35, 60, 61, 63, 76). More recently, the publicly accessible microbial genome databases were searched to reveal the presence of cphA-homologous genes in bacteria other than cyanobacteria, namely, Acinetobacter sp. strain ADP1, Bordetella bronchiseptica strain RB50, Bordetella pertussis strain Tohama I, Bordetella parapertussis strain 12822, Clostridium botulinum strain ATCC 3502, Nitrosomonas europaea strain ATCC 25978, and Desulfitobacterium hafniense strain DCB-2 (33, 80).

CGP is synthesized as membraneless granules, which are insoluble at physiological pH but soluble at high ionic strength and in acidic or alkaline conditions (34). However, a highly water-soluble CGP-like polymer has been produced in Escherichia coli BL21 expressing the cphA gene from D. hafniense (80). On the other hand, a more recent study revealed the possibility of obtaining both variants of CGP, i.e., soluble and insoluble CGP, irrespective of the E. coli host and the source of cphA (20). CGP is not susceptible to proteases (54, 62, 63); rather it is hydrolyzed intracellularly and extracellularly to Asp-Arg dimers by cyanophycinases (37, 45, 46, 51, 54). Purified CGP can be chemically converted to a derivative with reduced arginine content (30) or to completely biodegradable poly(aspartic acid) (5), which can be used as a substitute for nonbiodegradable polyacrylates with many technical and medical applications (5, 36, 52, 55, 59, 78).

Cyanophycin synthetases have been heterologously expressed in various heterotrophic bacteria such as Corynebacterium glutamicum, Ralstonia eutropha, Pseudomonas putida, and E. coli (4, 11, 24, 48, 79). Transgenic potato and tobacco plants expressing the cphA gene of Thermosynechococcus elongatus were recently also obtained; however, the plants accumulated only very little CGP in comparison to bacteria (42). Biotechnological production of CGP on a large scale was so far achieved in E. coli expressing cphA of Synechocystis sp. strain PCC6803. The cells accumulated CGP up to 24 or 28% (wt/wt) of cell dry matter (CDM), if they were cultivated in costly complex medium (19) or in protamylasse, which is a residue of industrial starch production from potatoes (18).

Several studies revealed the importance of aspartic acid and arginine provision in the medium as substrates for CGP synthesis (17, 66, 74). Arginine was also identified as the most important bottleneck for CGP production on a large scale using Acinetobacter sp. strain ADP1 (17). In an optimized mineral salts medium (MSM) supplemented with arginine as sole carbon source, cells of this bacterium accumulated CGP up to 46% of CDM, giving the highest cellular CGP contents ever reported (17). However, such high CGP contents were obtained only under phosphate limitation and if the MSM was supplemented with high concentrations of arginine (17). Therefore, engineering the pathways of arginine metabolism and their regulation may result in an improved metabolic flux towards provision of arginine in the bacterial cells. Both the metabolism and its regulation were studied in most detail in Pseudomonas aeruginosa (1, 26, 38, 50). Since the nutritionally versatile Acinetobacter sp. strain ADP1 is closely related to P. aeruginosa, and its recently released genome sequence is available (10), we decided to identify the relevant genes in this strain on the basis of similarities to the known genes of P. aeruginosa and other bacteria. This study applied various strategies to improve CGP biosynthesis in Acinetobacter sp. strain ADP1 by constructing mutants unable to utilize arginine as a carbon source, by disrupting a putative gene encoding the arginine regulatory protein, and by enhancing biosynthesis of glutamate as a precursor of arginine biosynthesis. In addition, the increase of CGP biosynthesis and its accumulation in the absence of phosphate-limiting cultivation conditions were addressed.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains, plasmids, DNA fragments, and cultivation conditions.
All bacterial strains and plasmids used in this study are listed in Table 1.


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  		TABLE 1. Strains and plasmids used in this study

 
Media and growth conditions.
The MSM used for cultivation of Acinetobacter sp. strain ADP1 contained 20 mM KCl; 0.8 mM MgSO4 · 7H2O; 50 µM of Na2HPO4 · 12H2O (unless specified otherwise); and per liter 6 mg of FeCl3 · 6H2O, 6 mg of MnCl2 · 4H2O, 6 mg of ZnCl2, 6 mg of CuSO4 · 5H2O, 6 mg of CaCl2 · 2H2O, and 6 mg of NaCl. MSM was supplemented with appropriate carbon and nitrogen sources as indicated in the text. For routine cultivation and transformation experiments, Acinetobacter sp. strain ADP1 was cultivated at 30°C in Luria-Bertani (LB) (56) medium. E. coli was grown at 37°C in LB medium. During all cultivations flasks were agitated on a shaker at 130 rpm with a horizontal amplitude of about 5 cm. Protamylasse was obtained from Avebe (Veendam, The Netherlands) and was prepared as described previously (18).

Determination of CDM.
For determination of CDM, an aliquot of a culture was centrifuged at 4°C in a bench centrifuge at 3,500 x g. The supernatant was carefully discarded. The cell pellet was washed after suspension in a saline solution (0.9% [wt/vol] NaCl) by centrifugation and lyophilized, and its mass was determined gravimetrically.

Purification and analysis of CGP.
CGP was isolated from the cells according to the procedure described previously (17). CGP was also extracted by the fast acid extraction method, and its concentration was determined according to the method of Bradford (14). The amino acid constituents of the isolated material were determined by high-pressure liquid chromatography (19).

Preparation of cell extracts and assay of cyanophycin synthetase activity.
The activity of cyanophycin synthetase in the soluble cell fraction was assayed by the radiometric method described previously (3).

Identification of genomic sequences for genes used in this study.
All genes studied from Acinetobacter sp. strain ADP1 were identified by similarity to the corresponding genes in P. aeruginosa strain PAO1 or other bacterial strains using the complete primary genome sequence from Genoscope and confirmed later after the genome sequence of the strain was published (10).

Isolation, manipulation, and transfer of DNA.
Genomic DNA was isolated from Acinetobacter sp. strain ADP1 by the procedure described in reference 53. Plasmid DNA was isolated from E. coli by the alkaline lysis method (12). Isolation of DNA fragments from an agarose gel and purification of PCR products were carried out using a Nucleo-trap kit (Macherey-Nagel, Düren, Germany). Restriction enzymes and other DNA-manipulating enzymes were used as described by the manufacturers. For transformation of E. coli Top10, competent cells were prepared by the CaCl2 method (25). Transformation of Acinetobacter sp. strain ADP1 was done as described previously (49). Cells were grown in 100 ml of LB medium at 30°C for 12 h, and 1 ml of this culture was then used to inoculate 24 ml of prewarmed LB medium and incubated for 2 h at 30°C with shaking. Five hundred microliters of this suspension was then incubated with 8 µg of linear DNA for 2 h at 30°C, and the cells were subsequently spread on LB agar plates containing appropriate concentrations of kanamycin or tetracycline.

Generation of astA and argR mutant.
All oligonucleotides used in this study are listed in Table 2. A DNA fragment comprising the astA gene (808 bp) was amplified by PCR from genomic DNA of Acinetobacter sp. strain ADP1 by using Platinum Pfx DNA polymerase (Invitrogen) and the primers PastI and PastII. The PCR product was directly ligated into SmaI-digested pBluescript SK– DNA (Stratagene). A recombinant plasmid (pYast) containing astA in a collinear orientation with respect to the lacZ promoter was chosen, digested with StuI, and then ligated with a blunt-ended kanamycin resistance cassette. A fragment flanked by astA sequences was isolated using the restriction enzymes Eco32I and BamHI and directly transformed into competent cells. PCR with primers PastI and PastII was employed to confirm the presence of a 1,788-bp fragment comprising astA plus the 980-bp kanamycin resistance cassette and the absence of a full-length 808-bp astA fragment in a kanamycin-resistant clone. For disruption of the putative argR gene, a DNA fragment containing the argR gene was amplified by standard PCR using Pfx DNA polymerase and the primers PargI and PargII to obtain a 708-bp fragment which was ligated into SmaI-digested pBluescript SK– DNA to obtain pYargR. The inverse PCR primers Pin-argI and Pin-argII were designed to remove a central 186-bp region from the putative argR gene. The resulting PCR product was isolated from a gel and ligated with a kanamycin resistance cassette. The resulting plasmid pYargR{Omega}Km was digested with Eco32I plus BamHI, and a 1,496-bp fragment was isolated and transformed into Acinetobacter sp. strain ADP1 to obtain the {Delta}argR mutant defective in the ability to form the arginine regulatory protein. The genotype of relevant clones was confirmed by PCR and restriction analysis.


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  		TABLE 2. Primers used in this study

 
Construction of argF-, carA-carB-, pepC-, and phoB-overexpressing strains of Acinetobacter calcoaceticus.
To construct plasmids providing extra copies of argF, carA-carB, pepC, and phoB, primers listed in Table 2 and Pfx DNA polymerase were used to amplify the corresponding DNA fragments from genomic DNA of Acinetobacter sp. strain ADP1. The genes were finally cloned into the 6.45-kbp plasmid pWH1274, which is a derivative of pBR322 containing the origin of replication of Acinetobacter sp. (27). For this, ligation mixtures were transferred into competent cells of E. coli Top10, and clones were selected on LB agar plates containing 12.5 µg/ml tetracycline. Plasmids were then extracted, examined for the presence and orientation of the inserted fragments, and subsequently transformed into naturally competent cells of Acinetobacter sp. strain ADP1. The ornithine carbamoyltransferase-encoding argF gene was amplified using primers PargFI and PargFII. The resulting 1,044-bp PCR fragment was then ligated to ScaI-digested pWH1274 to yield plasmid pYargF. Similarly, a 4,484-bp DNA fragment containing the carAB operon, encoding the small (CarA) and large (CarB) subunits of carbamoylphosphate synthetase, was cloned into the ScaI restriction site of pWH1274, yielding plasmid pYcarAB. Plasmid pYpepC was constructed by cloning a 2,887-bp DNA fragment containing pepC, which encodes the phosphoenolpyruvate carboxylase, after amplification of the gene using the primers PpepcI and PpepcII. The phoB gene belonging to the phosphate regulon was amplified by using the primers PphoBI and PphoBII. The resulting 736-bp fragment was ligated into ScaI-treated pWH1274 DNA to yield plasmid pYphoB.

Construction of Acinetobacter sp. strain ADP1 defective in astA and overexpressing the argF gene.
To construct the Acinetobacter sp. strain ADP1 defective in astA and overexpressing the argF gene, the A. calcoaceticus strain {Delta}astA constructed in this study was transformed with plasmid pYargF (Table 1).

Enzyme assays.
The ornithine carbamoyltransferase and carbamoylphosphate synthetase were determined as described previously (50), and the reaction product citrulline was determined colorimetrically (13). Phosphoenolpyruvate carboxylase (PepC) was assayed according to the method of Mori and Shiio (41). Alkaline phosphatase activity was determined according to the method of Garen and Levinthal (21). One unit of enzyme activity refers to the conversion of 1 µmol of substrate into product per minute.

All assays were determined with crude extracts obtained by sonication of cells chilled with ice. Protein concentrations were determined according to the method of Bradford (14).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of a cluster of genes coding for CGP metabolism and arginine catabolism in Acinetobacter sp. strain ADP1.
All genes coding for the enzymes of arginine biosynthesis were identified by similarity to those of other bacteria; they are distributed throughout the chromosome as in the genome of P. aeruginosa PAO. Genes coding for enzymes of the arginine succinyltransferase (AST) and arginine decarboxylase pathways of arginine utilization were shown to be present, whereas an arginine deiminase gene was lacking. A layout of all metabolic pathways of Acinetobacter sp. strain ADP1 is available at http://www.genome.ad.jp.

One interesting feature of this strain is the occurrence of a gene cluster encoding enzymes for CGP synthesis (cphA) and degradation (cphI) plus genes encoding enzymes of the AST pathway of arginine utilization (ast operon) and the arginine regulator protein (argR) as shown in Fig. 1. Such a gene cluster was not observed in any other CGP-synthesizing bacterium (data not shown). The arginine succinyltransferase (AstA) catalyzes the first reaction of this pathway by transferring a succinyl moiety to arginine (Fig. 2). The astA homologue in Acinetobacter sp. strain ADP1 exhibited up to 58% amino acid similarity to corresponding genes from other bacteria. The putative ArgR of Acinetobacter sp. strain ADP1 exhibited up to 54% amino acid similarity to other members of the AsnC/Lrp family of transcriptional regulators. There are two open reading frames between cphB and argR (Fig. 1): (i) HP2 exhibited 40% and 37% similarity to the succinylglutamate desuccinylase/aspartoacylase of Pseudomonas syringae and to the arginine and ornithine transport protein of P. aeruginosa PAO, respectively, and (ii) HP1 codes for a putative protein without any homology to any previously known sequence. On the other hand, two genes were found upstream of cphA (Fig. 1): HP3, encoding a putative amino acid transporter, and a homologue of the universal stress protein UspA. These two proteins probably play an important role relevant to cyanophycin biosynthesis and amino acid metabolism.


Figure 1
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  		FIG. 1. Organization of genes encoding enzymes of arginine catabolism and CGP metabolism in the genome of Acinetobacter sp. strain ADP1 (middle). The disruption of the astA gene encoding arginine N-succinyltransferase by insertion of a kanamycin resistance cassette is shown in the top part. Inactivation of the putative argR gene was obtained by deletion of a central 186-bp region and insertion of an {Omega}Km element (bottom). HP1 (ACIAD1281) codes for a putative protein showing no homology to any previously known sequence. HP2 (ACIAD1282) encodes a putative protein with high similarities to succinylglutamate desuccinylase/aspartoacylase and to arginine and ornithine transport protein. HP3 (ACIAD1278) encodes a putative amino acid transporter. Each 1 cm is equal to 1 kbp.

 

Figure 2
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  		FIG. 2. Putative pathway of arginine metabolism in Acinetobacter sp. strain ADP1. Only key metabolites and known genes are indicated. Dashed lines point to genes which are under the control of ArgR: (–), ArgR together with arginine represses the transcription of carAB and argF; (+), ArgR together with arginine induces the ast operon. Genes shown in boldface were overexpressed. The astA and argR genes were inactivated (x) in this study. TCA, tricarboxylic acid.

 
Influence of astA- and argR-homologous genes on CGP accumulation.
One goal of these experiments was to abolish the ability of the cells of Acinetobacter sp. strain ADP1 to utilize arginine. A second goal was to abolish the negative effect of ArgR on arginine biosynthesis. Therefore, the astA gene was inactivated by insertion of an {Omega}Km element as described in Materials and Methods. Similarly, the putative argR gene was inactivated by deletion and insertion of a {Omega}Km resistance cassette. Cells of the {Delta}astA mutant and of the {Delta}argR mutant were unable to grow in MSM supplemented with arginine as sole carbon source. Cells with inactivated astA were obviously unable to perform the first step of arginine utilization via the AST pathway, whereas cells defective in ArgR lacked the activation of the AST pathway when they were grown on arginine as sole carbon source. The capability of the two mutants to synthesize and accumulate CGP from different carbon sources in comparison to the wild type is shown in Table 3. Whereas cells of the wild type accumulated CGP to only 2% (wt/wt) of CDM when grown on sodium glutamate as a carbon source, cells of the {Delta}astA or the {Delta}argR mutant contained CGP at 3.9 or 5.5% (wt/wt) of CDM, i.e., at about a 2- and a 2.5-fold-higher level, respectively, when cells were grown for 44 h in 100 ml MSM with sodium glutamate as a carbon source. The wild type accumulated CGP up to 42% (wt/wt) of CDM when arginine was provided as sole carbon source. However, if sodium glutamate was added, the CGP content of cells was only 2.6% (wt/wt) of CDM. In contrast the reductions of the CGP contents of the cells were less severe in the {Delta}astA mutant and also in the {Delta}argR mutant, which accumulated CGP up to 17.9 or 9.5% (wt/wt) of CDM, respectively, when grown under the same conditions. The ability of the {Delta}astA and the {Delta}argR mutants to synthesize high CGP contents using arginine as a nitrogen source revealed that inactivation of argR or astA did not affect the ability of the two mutants to be transported into the cells and to incorporate arginine into the CGP polymer.


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  		TABLE 3. CGP biosynthesis and accumulation in the wild type and {Delta}astA and {Delta}argR mutants of Acinetobacter sp. strain ADP1 during cultivation on different carbon sourcesf

 
As shown in Table 4, cells of the wild type and of the {Delta}astA and {Delta}argR mutants could utilize sodium glutamate as sole source for carbon, nitrogen, and energy for growth. Interestingly, the {Delta}argR mutant synthesized significantly more CGP than the wild type and the {Delta}astA mutant. CGP accumulation was much more stimulated by addition of arginine to the medium in the {Delta}astA and in particular in the {Delta}argR mutant. Interestingly, the wild type accumulated more CGP (18.8% of CDM) in comparison to the {Delta}astA and {Delta}argR mutants (0.3 and 4.3%, wt/wt, of CDM, respectively) when cultivated on sodium citrate as a carbon source and arginine as a nitrogen source for 30 h in 50 ml MSM. Conversely, all strains accumulated the same low amounts of CGP (about 1.2 to 1.7% of CDM) in the presence of sodium citrate plus ammonium chloride and in the absence of arginine, thus revealing the importance of arginine addition especially during cultivation on intermediates of the citric acid cycle such as citrate.


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  		TABLE 4. CGP biosynthesis and accumulation in the wild type and {Delta}astA and {Delta}argR mutants of Acinetobacter sp. strain ADP1g

 
Biosynthesis and accumulation of CGP in the {Delta}argR mutant were also studied in relation to the concentrations of sodium glutamate and ammonium chloride. In addition, the ratio of medium volume to flask volume was varied (data not shown). Increasing the concentration of glutamate as a carbon source exerted a slightly positive effect on CGP accumulation, and in the presence of 110 mM sodium glutamate the cells accumulated about 1.5- to 2.0-fold more CGP than in the presence of only 25 mM sodium glutamate. In contrast, increasing the concentration of ammonium chloride (from 10 to 50 mM) or decreasing the medium volume (from 100 to 50 ml) in relation to flask volume (fixed at 250 ml) exerted a slightly negative effect on CGP accumulation.

Overexpression of ArgF, CarA-CarB, and PepC.
In other bacteria such as P. aeruginosa and E. coli expression of argF encoding ornithine carbamoyltransferase and the carAB operon encoding the small (CarA) and the large (CarB) subunits of carbamoylphosphate synthetase is repressed by ArgR and arginine (29, 38, 73). Since little is known about the regulation of arginine metabolism in Acinetobacter sp. strain ADP1, we attempted to overexpress the enzymes mentioned above (shown in boldface in Fig. 2) in order to abolish the repression caused by ArgR and to enhance arginine biosynthesis and consequently CGP biosynthesis if carbon sources other than arginine are used. Since CGP biosynthesis in cells of Acinetobacter sp. strain ADP1 using sugars or other carbohydrates as substrates gave only low amounts of CGP and since it was recently reported that Acinetobacter sp. strain ADP1 is lacking a gene encoding pyruvate kinase, which is involved in the conversion of carbohydrates to pyruvate (10), the phosphoenolpyruvate carboxylase encoded by pepC catalyzing the conversion of phosphoenolpyruvate to oxaloacetate as one of the anaplerotic reactions was also overexpressed to augment the utilization of carbohydrates and to ultimately increase the metabolic flux towards 2-oxoglutarate, which is the substrate for glutamate and subsequently arginine biosynthesis.

Cells overexpressing ArgF contained about three times more CGP than did cells expressing CarA-CarB or PepC when grown on LB complex medium (Table 5) and about four times more CGP than the wild type (Table 3). From sodium gluconate as a carbon source for CGP synthesis either strain accumulated about as little CGP as the wild type (Table 5). Otherwise, cells of all recombinant strains showed significantly higher CGP contents than the wild type when sodium glutamate was used as sole source of carbon (Table 5). In particular the strain harboring pYargF accumulated about 5.5-fold more CGP than the wild type did, thus indicating an important role of ornithine carbamoyltransferase (ArgF) for CGP synthesis in the absence of arginine from the medium. If the recombinant strains harboring pYargF, pYcarAB, or pYpepC were grown in MSM containing arginine as a carbon source, the CGP contents of the cells were high and ranged from 15.3 to 40.9% (wt/wt) of CDM. However, no higher CGP contents than those with the wild type were obtained. Overexpression of argF, carAB, or pepC also exerted a positive effect on CGP accumulation during cultivation of the cells in the presence of aspartic acid; in comparison to the wild type the CGP contents of the cells increased by a factor of about 3.6, 1.8, or 1.6, respectively (Tables 3 and 5). During cultivation on fructose, glucose, or molasses these recombinant strains did not exhibit significant increases of the CGP contents of the cells in comparison to the wild type (data not shown). With respect to the impact on CGP contents of the cells, the substrates could be arranged in the following order: arginine > glutamate > aspartic acid > gluconate ≥ LB.


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  		TABLE 5. CGP biosynthesis and accumulation in recombinant strains of Acinetobacter sp. strain ADP1 harboring the plasmid pYargF, pYcarAB, or pYpepC, when grown on different carbon sourcesd

 
Specific activities of the overexpressed enzymes were determined in the wild type and the recombinant strains. Specific activity of ornithine carbamoyltransferase was 26% higher in the strain harboring pYargF (134.2 ± 5.3 mU/mg protein) than in the wild type (106.1 ± 3.2 mU/mg protein). On the other hand, the activity of carbamoylphosphate synthetase was increased 3.6-fold in cells of the strain harboring pYcarAB (11.6 ± 0.7 mU/mg protein) in comparison to the wild type (3.2 ± 0.6 mU/mg protein). Cells of the recombinant strain harboring pYpepC showed 80% more activity of phosphoenolpyruvate carboxylase (16.5 ± 0.9 mU/mg protein) than did the wild type (9.2 ± 1.2 mU/mg protein). Therefore, increases of CGP contents in the recombinant cells expressing the different genes paralleled the increase of specific activities of the corresponding enzymes.

A recombinant strain with an inactivated astA gene, which contained pYargF, synthesized and accumulated significantly high amounts of CGP compared to all strains investigated so far when cultivated in LB medium or in the cheap protamylasse. The Acinetobacter sp. strain ADP1 {Delta}astA (pYargF) accumulated 29.3% ± 0.3% and 25.5% ± 2.7% (wt/wt) of CDM when grown in 2% (vol/vol) protamylasse and LB, respectively. Cells were cultivated in 250-ml Erlenmeyer flasks containing 100 ml LB or 2% protamylasse supplemented with 50 µg/ml kanamycin and 12.5 µg/ml tetracycline, incubated at 30°C for 24 h with shaking. There were 1.6- or 1.3-fold increases of the specific activity of ornithine carbamoyltransferase in cells of the mutant harboring pYargF. The bottleneck of arginine provision is solved using this recombinant strain: cells of Acinetobacter sp. strain ADP1 {Delta}astA (pYargF) grown on the residual waste protamylasse (without additional arginine) accumulated up to 75% of the CGP accumulated by Acinetobacter sp. strain ADP1 grown on arginine as sole source of carbon. Therefore, reduction in cultivation costs and high contents of CGP in a strain of Acinetobacter sp., ADP1, were achieved without arginine provision.

Overexpression of PhoB.
In order to study the effect of phosphate limitation and to allow cells of Acinetobacter sp. strain ADP1 to accumulate CGP also in medium containing nonlimiting concentrations of phosphate, the genome sequence of Acinetobacter sp. strain ADP1 was also investigated for a phoB gene encoding a positive activator (PhoB) of the phosphate regulon. The putative phoB-containing DNA fragment was amplified and cloned as described in Materials and Methods. Cells of strain ADP1 harboring pYphoB providing an extra copy of phoB were cultivated in MSM containing sodium glutamate or arginine as sources for carbon in the presence of three different concentrations of phosphate. The CGP contents of the cells were analyzed and compared to those obtained for cells of the wild type, which were cultivated under identical conditions (Table 6). The results show that under conditions of severe phosphate limitation (42 µM) both strains synthesized and accumulated about the same amounts of CGP from glutamate or arginine. However, at less severe or nonlimiting concentrations of phosphate (0.16 or 1.3 mM, respectively), cells of the recombinant strain harboring pYphoB accumulated about 1.8- or 8.6-fold more CGP than cells of the wild type, yielding CGP contents of the cells of 20.8 or 4.3% (wt/wt) of CDM, respectively. CGP accumulation from glutamic acid was again very low and not affected by plasmid pYphoB (Table 6).


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  		TABLE 6. Effect of the overexpression of PhoB protein in cells of Acinetobacter sp. strain ADP1 on biosynthesis and accumulation of CGP during cultivation at different phosphate concentrations in the presence of glutamate or argininea

 
The previous data were confirmed by the estimation of the activity of alkaline phosphatase. Cells of the wild type previously grown at a phosphate surplus (1.3 mM) showed a lower specific activity (0.35 ± 0.08 mU/mg protein) than did cells grown in phosphate-limited (42 µM) medium (1.68 ± 0.11 mU/mg protein). However, Acinetobacter sp. strain ADP1 harboring pYphoB showed a significant increase in the specific activity of the phosphatase (2.76 ± 0.04 mU/mg protein) when cultivated under phosphate starvation and showed an activity of 0.5 ± 0.03 mU/mg protein when cultivated at 1.3 mM phosphate.

Cyanophycin synthetase activity.
The analysis of cells of Acinetobacter sp. strain ADP1 revealed high specific activities of cyanophycin synthetase. The activity was in particular high in cells grown on arginine (23.3 U/mg protein) that had accumulated up to 40% (wt/wt) CGP of CDM. The specific activity was slightly lower but still very high in cells grown on glutamate (14.4 U/mg protein), in which CGP accounted for only 1.3% (wt/wt) of CDM. The increase in alkaline phosphatase activity under conditions of phosphate limitation and in strains containing an extra copy of phoB may indicate a role of PhoB protein in activation of cphA transcription under phosphate starvation or when PhoB is overexpressed beside the availability of arginine as an essential substrate for CGP accumulation.

Properties of the accumulated CGP.
CGP samples obtained from any of the investigated strains and from any condition investigated in this study contained typically only aspartic acid and arginine; analysis of CGP samples by high-pressure liquid chromatography showed that no other amino acids were detected as constituents. Furthermore, the molecular masses of the accumulated CGP were in the same range as described previously (17, 33).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Acinetobacter sp. strain ADP1 was the first noncyanobacterial strain shown to synthesize CGP. The aim of this study was to reveal molecular factors which affect synthesis of CGP in this bacterium. Synthesis of CGP in A. calcoaceticus strain ADP1 depends on two crucial factors: (i) phosphate limitation and (ii) provision of arginine. CGP amounts of up to 46% of cell dry matter were obtained when wild-type cells were grown in the presence of arginine as sole source of carbon (17). In this study the approach was made to expand CGP synthesis using sources for carbon other than arginine by disruption of genes involved in arginine utilization and regulation or by overexpression of genes coding for enzymes relevant in arginine biosynthesis. Moreover, regulation of phosphate starvation was also considered. The presence of cphA and cphI encoding cyanophycin synthetase and cyanophycinase, respectively, together with genes of the ast operon encoding enzymes responsible for arginine utilization as a carbon source in the same cluster indicates the importance of CGP as a reserve material under starvation conditions. Cells of A. calcoaceticus strain ADP1, which had previously accumulated CGP, were capable of degrading this storage compound upon the transfer of the cells into medium containing no carbon source (unpublished data). The products of intracellular CGP degradation could be arginine and aspartic acid, Asp-Arg dipeptide, or basically free arginine plus about 30% dipeptide (9, 23, 60). The ast operon could, therefore, be induced by the regulator ArgR mediated by arginine which is released from degradation of CGP. Growth experiments with A. calcoaceticus strain ADP1 in MSM supplemented with only arginine showed that this strain utilizes arginine as a source of carbon, nitrogen, and energy (data not shown).

In most bacterial strains where regulation of arginine metabolism has been investigated, transcriptional repression of anabolic reaction and induction of catabolic pathways proved to be exerted by the binding of arginine to the arginine regulatory protein (ArgR) (38, 39, 64, 71). ArgR proteins from E. coli, Bacillus stearothermophilus, and Bacillus subtilis belong to the protein family of helix-turn-helix DNA binding proteins (43, 67, 72, 77), whereas ArgR from P. aeruginosa is of a different type; it possesses two helix-turn-helix domains and belongs to the AraC/XylS family (50). The argR homologue from Acinetobacter sp. strain ADP1 exhibited similarities to various proteins belonging to the AsnC/Lrp protein family. In Acinetobacter sp. strain ADP1 (Fig. 1), the AsnC/Lrp regulators were found adjacent to the target genes as in Yersinia pestis, Haemophilus influenzae, and Salmonella enterica serovar Typhimurium (15). It was shown that these proteins are involved in regulation of amino acid metabolism: anabolic reactions were down-regulated in the presence of the respective amino acids and catabolic reactions were up-regulated in their presence (15).

A. calcoaceticus strain ADP1 showed a similar behavior, and the highest amounts of CGP were synthesized and accumulated using arginine as sole source for carbon and a low phosphate concentration. In this case, cells were capable due to arginine-mediated activation of the AST pathway for utilization of arginine as a carbon source. On the other hand, the anabolic pathway was repressed; therefore, the {Delta}astA mutant could not utilize arginine as a carbon source but as a nitrogen source, and the intracellular pool of arginine was probably increased as indicated by a 75%-higher CGP content of the cells in comparison to the wild type after growth on glutamate. Similarly, the {Delta}argR mutant was unable to grow on arginine as sole carbon source, confirming that ArgR activates the AST pathway (Fig. 2) and possibly expression of other enzymes involved in arginine utilization. In P. aeruginosa (26) ArgR together with arginine mediates induction of gdhB, which encodes glutamate dehydrogenase catalyzing conversion of glutamate into 2-oxoglutaric acid. These results explain the inability of Acinetobacter sp. strain ADP1 to synthesize significant amounts of CGP if the cells were grown on arginine and glutamate together as carbon and nitrogen sources. The increased accumulation of CGP by the {Delta}astA and {Delta}argR mutants of Acinetobacter sp. strain ADP1 was most likely due to the inability of the cells to utilize arginine for CGP synthesis or due to the lack of activation of gdhB as in the case of A. calcoaceticus strain {Delta}argR.

Synthesis of a CGP content of 18% of CDM in cells of Acinetobacter sp. strain ADP1 cultivated in the presence of citric acid plus arginine is consistent with findings in P. aeruginosa. In the latter it was shown that ArgR is subjected to catabolite repression by intermediates of the tricarboxylic acid cycle (44). The {Delta}astA and also the {Delta}argR mutant of Acinetobacter sp. strain ADP1 showed a significant reduction in CGP accumulation under previous conditions, where both strains are unable to utilize arginine as a carbon source. All investigated strains synthesized only little CGP when grown on citric acid in the absence of arginine (Table 4). Interestingly, cells of the {Delta}argR mutant were shown to contain multiple granules per cell (data not shown), whereas normally only one single large CGP granule is seen in cells of the wild type when grown on arginine (17).

Synthesis of CarA-CarB and ArgF is repressed by ArgR (2, 28, 29, 38, 73). When the homologous genes were overexpressed as in Acinetobacter sp. strain ADP1 harboring pYcarAB as indicated by a 3.6-fold-higher carbamoylphosphate synthetase activity than in the parent strain, the cells grew slowly only on glutamate as a carbon source; however, they accumulated more CGP. The CarA-CarB proteins catalyze synthesis of carbamoylphosphate; nevertheless, as in the wild-type strain the argF gene is still repressed or the enzyme is not sufficiently active to direct the metabolic flux towards arginine biosynthesis. Cells of Acinetobacter sp. strain ADP1 harboring pYargF synthesized relatively high amounts of CGP in comparison to the wild-type cells, although there was only a 28% increase of carbamoylphosphate transferase activity, revealing the importance of ArgF as a key enzyme in arginine biosynthesis. Cells of the Acinetobacter sp. {Delta}astA mutant harboring pYargF accumulated about 21-fold more CGP than all other investigated strains when cultivated on LB medium. In addition, this strain accumulated as much CGP as E. coli DH1(pMa/c5-914::cphA6803), which was cultivated in 6% (vol/vol) protamylasse (18). Therefore, the engineering of some specific genes of the arginine metabolism of Acinetobacter sp. strain ADP1 yielded strains with improved arginine biosynthesis as indirectly indicated by higher CGP contents of the cells.

Acinetobacter sp. strain ADP1 utilizes glucose as sole carbon source for growth, although genes coding for the glucose transporter phosphotransferase system and glucokinase were not detected (10). Previous biochemical studies (68-70) showed that glucose is catabolized exclusively via the Entner-Doudoroff (ED) pathway. This organism oxidizes glucose in the periplasm by the conversion of membrane-bound glucose dehydrogenase to gluconic acid, which accumulates transiently in the growth medium before it is metabolized by the ED pathway (69). Overexpression of phosphoenolpyruvate carboxylase (PepC) did not result in increased levels of CGP contents when cells were grown on glucose or gluconate. Only when the cells were grown on glutamate or aspartic acid as carbon source were high CGP contents obtained, albeit the activity of PepC was increased by 80%. These findings are consistent with data obtained from analysis of the Acinetobacter sp. strain ADP1 genome, which largely lacks genes for carbohydrate metabolism (10). The lower part of the ED pathway is hindered by the lack of pyruvate kinase. Additionally, 38% of the ABC transporters in Acinetobacter sp. strain ADP1 have been predicted to be involved in amino acid transport (10), also indicating that this strain preferably utilizes amino acids rather than sugars and carbohydrates.

Phosphate starvation triggers imperative metabolic and adaptive responses in several bacterial strains. Phosphate starvation was shown to induce poly(3-hydroxybutyrate) and CGP biosynthesis in Acinetobacter strains RA3849 (57) and ADP1 (33). The significant increase of CGP content in cells of a recombinant strain harboring plasmid pYphoB when cultivated in MSM, which contained arginine as sole source for carbon and a high phosphate concentration, provides evidence for the importance of the phoB gene. Expression of a majority of genes belonging to the phosphate regulon in E. coli is controlled by the PhoB/PhoR system, in response to inorganic phosphate levels (32, 40, 75). This system is composed of a response regulator (PhoB) and a sensor kinase (PhoR) for which homologues were identified in many other bacteria (31). The phoB or phoR genes of Acinetobacter sp. strain ADP1 were not investigated in detail previously; however, it was shown that polyphosphate kinase encoded by ppk was induced 5- to 15-fold during phosphate starvation (22). Expression of phoB from Acinetobacter sp. strain ADP1 harboring plasmid pYphoB resulted in a 42% increase of the specific activity of alkaline phosphatase and in an 8.6-fold increase of CGP content even at nonlimiting phosphate concentrations (1.3 mM). This indicates a role of PhoB in the enhancement of cphA transcription leading to increased CGP biosynthesis in comparison to the wild-type cells under the same cultivation conditions.


   ACKNOWLEDGMENTS
 
Yasser Elbahloul is indebted to the Deutsche Akademischer Austauschdienst and the government of the Arab Republic of Egypt for providing a fellowship.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Corrensstrasse 3, D-48149 Münster, Germany. Phone: 49 (251) 8339821. Fax: 49 (251) 8338388. E-mail: steinbu{at}uni-muenster.de. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, February 2006, p. 1410-1419, Vol. 72, No. 2
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