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Identification of genes affecting alginate biosynthesis in Pseudomonas fluorescens by screening a transposon insertion library

Abstract

Background

Polysaccharides often are necessary components of bacterial biofilms and capsules. Production of these biopolymers constitutes a drain on key components in the central carbon metabolism, but so far little is known concerning if and how the cells divide their resources between cell growth and production of exopolysaccharides. Alginate is an industrially important linear polysaccharide synthesized from fructose 6-phosphate by several bacterial species. The aim of this study was to identify genes that are necessary for obtaining a normal level of alginate production in alginate-producing Pseudomonas fluorescens.

Results

Polysaccharide biosynthesis is costly, since it utilizes nucleotide sugars and sequesters carbon. Consequently, transcription of the genes necessary for polysaccharide biosynthesis is usually tightly regulated. In this study we used an engineered P. fluorescens SBW25 derivative where all genes encoding the proteins needed for biosynthesis of alginate from fructose 6-phosphate and export of the polymer are expressed from inducible Pm promoters. In this way we would avoid identification of genes merely involved in regulating the expression of the alginate biosynthetic genes. The engineered strain was subjected to random transposon mutagenesis and a library of about 11500 mutants was screened for strains with altered alginate production. Identified inactivated genes were mainly found to encode proteins involved in metabolic pathways related to uptake and utilization of carbon, nitrogen and phosphor sources, biosynthesis of purine and tryptophan and peptidoglycan recycling.

Conclusions

The majority of the identified mutants resulted in diminished alginate biosynthesis while cell yield in most cases were less affected. In some cases, however, a higher final cell yield were measured. The data indicate that when the supplies of fructose 6-phosphate or GTP are diminished, less alginate is produced. This should be taken into account when bacterial strains are designed for industrial polysaccharide production.

Background

Linear polysaccharides composed of mannuronic and guluronic acid residues that may be O-acetylated, are denoted alginate. These polymers are synthesized by brown and some red algae and by bacterial species belonging to the genera Azotobacter and Pseudomonas. Alginates manufactured from brown algae are currently used in diverse industrial and pharmaceutical applications. However, alginates produced by bacteria can more easily be tailored to obtain the compositions desired for the more high-value end of the alginate market [1], and this has motivated our studies on alginate-producing bacteria.

Production of a secreted polysaccharide imposes a drain on the cell’s carbon and energy sources, and thus the biosynthesis is usually tightly regulated under natural conditions. In batch cultures, alginate-producing P. fluorescens mutants display a reduced cell yield compared to the corresponding non-alginate producing strains [2]. Bacterial alginate production is controlled by the alternative sigma factor AlgU and is usually turned off in Pseudomonas spp. Induction of alginate biosynthesis results in a proteolytic cascade that finally cleaves the AlgU anti-sigma factor MucA, leading to transcription of the genes in the alg operon [3].

In the first steps of bacterial alginate biosynthesis fructose 6-phosphate (Fru6P) is converted to GDP-mannuronic acid by the concerted action of AlgA, AlgC and AlgD. GDP-mannuronic acid is then polymerized to polymannuronic acid by Alg8 and the copolymerase Alg44. Together with AlgG, AlgX, AlgK and AlgE these form a protein complex that transports the alginate out of the cell as depicted in Fig. 1a [4]. AlgG also epimerizes some M-residues to G, while AlgI, AlgJ, AlgF and AlgX are needed to O-acetylate some of the M-residues. The alginate lyase AlgL removes alginate molecules that have been released to the periplasm [5]. Twelve of the thirteen genes directly involved in alginate biosynthesis are found in the alg operon, while the last, algC, is found elsewhere on the chromosome. This gene organization is found in all characterized alginate-producing bacteria. In addition to Fru6P and GTP, dimeric cyclic di-GMP (c-di-GMP) is needed for bacterial alginate biosynthesis [6, 7].

Fig. 1
figure1

The relationship between alginate biosynthesis and the cellular metabolism in P. fluorescens. a The proteins and metabolites needed for alginate biosynthesis. b A simplified model of the cell’s metabolism highlighting the processes identified in the present study as being important for full alginate biosynthesis levels. The genes discussed in the paper are highlighted in yellow. The Entner-Doudoroff pathway and the oxidative part of the pentose phosphate pathway are indicated by red arrows, and the non-oxidative part of the pentose phosphate pathway with purple arrows. Green arrows indicate other pathways competing with accumulation of the three metabolites Fru6P, GTP and c-di-GMP, while blue arrows indicate pathways that would increase the synthesis of one of these three metabolites. Each arrow may represent several enzymatic steps. Abbreviations: OM: Outer membrane, IM: Inner membrane, M: mannuronic acid residue, G: guluronic acid residue, Ac: Acetyl, TCA: Tricarboxylic acid cycle, PP: the non-oxidative part of the pentose phosphate pathway, GN6P: Glucosamine 6-phosphate, PG: Peptidoglycan, G6P: Glucose 6-phosphate, 6PG: 6-phosphogluconate, Pyr: Pyruvate, ILV: Isoleucine Leucine Valine, B5: Pantothenate, Trp: Tryptophan, PRPP: Phosphoribosyl pyrophosphate, R5P: Ribose 5-phosphate, E4P: Erythrose 4-phosphate

Recently we showed that the alginate synthesis rate is not proportional to the number of alginate biosynthetic complexes, indicating that there must be some kind of metabolic control as well [4]. In a recent transposon screen, some genes affecting AlgU-regulation were identified in P. aeruginosa [8]. However, the aim of the present study was to identify genes and pathways that influence alginate biosynthesis indirectly by perturbing the cell’s metabolism. An alginate-producing P. fluorescens strain in which the alg operon and algC is under control of the inducible Pm promoter was constructed and subjected to transposon mutagenesis. The Pm promoter and its activator XylS originally controls expression of the genes of the meta-cleavage pathway of aromatic hydrocarbons on the Pseudomonas putida plasmid pWW0 [9]. We have earlier shown that the Pm promoter and the weaker Pm promoter derivative Pm-G5 are useful for obtaining different levels of controlled gene expression in P. fluorescens [5]. About 11500 insertion mutants were screened with respect to growth and alginate biosynthesis, and the inactivated genes in mutants displaying altered alginate yields were identified. The results supported our hypothesis that further levels of post-translational regulation exist, allowing the cell to prioritize basic cellular metabolism over alginate biosynthesis.

Results and discussion

Construction of a P. fluorescens strain in which the alginate biosynthesis genes are controlled by the inducible Pm promoter

In order to avoid re-identification of the genes already known to directly regulate expression of the structural alginate biosynthetic genes, a derivative of P. fluorescens SBW25 designated strain MS1 was constructed (Fig. 2a). In this strain the naturally regulated algD promoter (which controls expression of the alg operon) was substituted with the wild-type Pm promoter. xylS, encoding the activator protein needed for expression from the Pm promoter, was inserted upstream of Pm. Then algC was inactivated by an in-frame deletion followed by a chromosomal insertion of a transposon containing a new algC copy expressed from a mutant version of Pm (PmG5) [5, 10]. This strain, designated MS2, produces only a small amount of alginate in the absence of Pm induction due to the low uninduced activity of PmG5.

Fig. 2
figure2

Genotypes for selected genetic constructs used in this study. a Strain MS1 in which the Pm promoter and the gene encoding XylS is inserted between the promoter and start codon of algD. b Strain MS2 in which a transposon expressing algC from PmG5 is inserted into PFLU2944 in an algC derivative of MS1. c Map of the transposon TnMS11 used for mutagenesis in this study. d Strain HE230 in which the gene encoding XylS and the PmG5 promoter is inserted between the promoter and start codon of algC in SBW25mucA. Inactivation of mucA confers a high level expression from wild type PalgD. Relevant promoters, and the two restriction sites used for sequencing are displayed above each map-line. The alg-genes are coloured to match Fig. 1, other P. fluorescens genes flanking the genes of interest are coloured blue, and heterologous genes and elements are coloured green. I and O denote the minitransposon ends

Alginate production has been reported to affect cell yield in P. fluorescens [2], and it was also possible that m-toluic acid would have an effect on growth. This was tested by cultivating the non-alginate producing wild type strain SBW25 and strain MS2 in Biolector® for three days in 0.5 x PIA supplemented with glycerol as carbon source. Growth rate and cell yield was significantly lower for the induced strain MS2 relative to the non-alginate producing strain, while no effect was seen by cultivating SBW25 in the presence or absence of 0.5 mM m-toluic acid (Additional file 1: Figure S1).

The transposon carrying algC was found to disrupt PFLU2944, which is the last gene in an operon encoding a putative ABC transporter (Fig. 2b). In the presence of the Pm/PmG5 inducer (m-toluate), the alginate production of strain MS2 was similar to that of strain MS1 (results not shown).

Construction of a transposon insertion library and screening with respect to alginate synthesis

The transposon-containing suicide vector pMS11 (Fig. 2c) was used for mutagenesis of strain MS2. Nearly 11500 insertion mutants were picked robotically from the original agar medium plates and cultivated in 96-deep-well microtiter plates containing 0.5x liquid PIA with glycerol and m-toluate. After three days, cell densities and alginate production were measured. The initial screen was followed by a rescreen of primary candidates and 184 mutants were found to produce less than 50% (163 mutants) or more than 110% alginate (21 mutants) when compared to the parent strain. The transposon insertion sites in all these mutants were determined by DNA sequencing, leading to identification of 134 different genes belonging to most of the main cellular functions (results not shown). Of these genes only ten were known alginate biosynthesis structural genes, while one was xylS, the positive regulator of Pm expression. These results show that about 92% of the identified genes are not directly associated with alginate synthesis. The screen did not cover all relevant genes in the genome, since insertions in algG, algF and algI (members of the alg operon) were not found.

Evaluation of the mutants to select candidates for further studies

Sequenced mutants with altered alginate phenotypes were cultivated in triplicates in 96-deep-well microtiter plates in three different media; 0.5xPIA with glycerol and 0.5xDEF4 with fructose or glycerol as carbon sources (7 g/L), and 0.5 mM m-toluate to induce alginate production. In the DEF4 media ammonium is the only nitrogen source, while PIA contains peptone that may be used as both nitrogen and carbon source. Furthermore, DEF4 contains more phosphate than PIA. The alginate yield from the control strain (MS2) was significantly higher in the DEF4 media, about 3 g/L compared to about 1 g/L in PIA, which resulted in better accuracy of the data in DEF4 for low alginate producers.

Results for mutants displaying significantly altered alginate production levels in at least one of the three media, are shown in Table 1. Significant changes were defined as less than 50% or more than 110% of the alginate production of the parent strain, and 36% of the retested gene-inactivation mutants did not meet this criterion. No mutant produced more alginate than the control strain in all three media. Mutants with insertions in alginate biosynthetic genes and xylS did, as expected, not produce alginate and are not included in Table 1. When several mutants had the same gene inactivated and displayed similar phenotypes, results from only one of them are shown in Table 1. For mutants where genes involved in glycerol utilization, amino acid biosynthesis or phosphate uptake had been inactivated, one would expect that the observed effects on biomass and alginate yield should be media dependent. As shown in Table 1 this was the case for most genes belonging to these categories.

Table 1 Identified mutants and their growth yield and alginate production in the three mediaa

It is probable that in many cases the phenotype observed in a transposon insertion mutant is caused directly by inactivation of the identified gene. However, polar effects (particularly in operons) and unrelated, spontaneous mutations can certainly not be excluded. For those genes where several independent transposon insertion mutants were identified, it is more likely that the observed phenotype is caused by the observed transposon insertion. The same argument may be used when several genes encoding proteins in the same metabolic pathway have been identified. In addition, 18 of the identified genes were chosen to be complemented either by expressing the wild type gene on a transposon or by adding the lacking metabolite. The transposons were constructed and transferred to the mutant strains, and both the mutant strains and the complemented strains were cultivated in two new growth experiments (Tables 2 and 3). Two of the 18 mutants could not be complemented and are not discussed further. These results show that the phenotypes of 16 out of 18 (89%) tested mutants can be explained by the transposon insertions only.

Table 2 Growth and alginate production of mutants using medium supplements or complementing transposonsa
Table 3 Effect of PhoBR disruptions on P. fluorescens growth and alginate biosynthesis

Alginate biosynthesis requires a functional biosynthetic complex, Fru6P and a dimeric form of c-di-GMP (Fig. 1a). Interestingly, the majority of those mutants that reproducibly produced less alginate were assigned to the groups involved in uptake and metabolism of carbohydrates, amino acids and nucleotides (Table 1). In addition four genes encoding proteins involved in protein modification were identified. Fig. 1b summarizes how the pathways identified in the current study might influence alginate yield, and these genes and pathways are discussed in more detail below.

Alginate production is influenced by signal transduction systems involved in carbon, nitrogen and phosphor metabolism

Four different signal transduction systems, CbrAB, NtrBC, PTSNtr, and PhoBR, were identified in the screen by using the criteria of either complementation or identification of several independent mutants in specific genes or pathways. The CbrAB two-component system has been described in several species of Pseudomonas as sensors and regulators of genes involved in utilization of different carbon and nitrogen sources, and has been proposed as sensors for the C/N balance in the cell [11, 12]. It has been shown that CbrB activates the expression of non-coding RNAs that relieve the catabolite repression otherwise exerted by Crc [13]. In P. putida, inactivation of cbrB also affected stress responses and biofilm development [14]. Our results show that the identified cbrB mutant produces less alginate (0-63%) than the otherwise isogenic control strain in all three media (Table 2). The mutant could be complemented by introducing a transposon-encoded copy of cbrB (Table 2). The effect of inactivating cbrA was, however, less pronounced, and might be caused by a polar effect on cbrB (Table 1). In P. putida, a cbrB mutant was shown to be unable to use some amino acids as carbon source, and to have an increased expression level of some of the genes encoding proteins involved in the Entner-Doudoroff pathway [14]. If the consequences of inactivating cbrB is similar in P. fluorescens, these two effects alone might explain the observed growth and alginate yields for the cbrB mutants, by reducing the net flow to Fru6P (Fig. 1b). However, given the known pleiotropic nature of a cbrB mutation, this probably is not the full explanation.

NtrBC is known to be an important response regulator system for bacterial nitrogen sensing, and has been found to interact with the CbrAB system [14]. GlnE is needed for the posttranscriptional activation of glutamine synthase, which is a part of the NtrC regulatory cascade [15]. It has been shown that inactivation of this gene lowered the pool of Fru6P in Corynebacterium glutamicum [16]. Consistent with this the alginate yield was significantly lower in PIA and in DEF4 with fructose for both glnE mutants (Table 1).

Glutamine and α-ketoglutarate are used by the NtrC-cascade to sense the carbon and nitrogen status of the cell, and these metabolites were recently found to affect the phosphorylation rate of the nitrogen-related phosphoenolpyruvate phosphotransferase system (PTSNtr) in E. coli [17]. PTSNtr is also known to form a link between carbon and nitrogen metabolism in pseudomonads [18]. While fructose is probably imported and phosphorylated by a PTS in P. fluorescens, glycerol is taken up through a transport and kinase system and is fed into the central metabolism as triose phosphates [19]. PtsP (EINtr) is the first protein in the nitrogen-related phosphate relay, and the two ptsP mutants identified in the current study produced low amounts of alginate both in PIA (24 and 8%) and in DEF4 with fructose (14 and 5%). An earlier study has shown that a ptsP mutant of P. putida produces less polyhydroxyalkanoate than the wild type, and it was suggested that such a mutant would behave as if there was a carbon limitation [20]. A similar argument could be used to explain the lower yield of alginate in our ptsP mutant. Recently it was also shown that inactivation of ptsP in P. aeruginosa decreases the level of c-di-GMP [21].

The response regulator PhoB and the histidine kinase PhoR control the Pho-regulon, which covers a major pathway for bacterial adaptation to phosphate starvation. PhoB may also be activated by other kinases [22]. Since phoB and phoR form an operon, new in-frame deletion mutants for each of these genes were constructed in the alginate-producing strain SBW25mucAHE230 (Fig. 2d). This strain was chosen because our standard gene recombination vector could not be used in the tetracycline-resistant strain MS2. The wild-type genes were cloned both individually and as an operon on transposons, and these transposons were used to complement the deletion mutants. The new phoR mutant behaved similarly to the wild type strain, while the phoB deletion resulted in lower cell yield and no alginate production when cultivated in DEF3 with reduced phosphate concentration (1 μM) (Table 3). Both traits were restored by chromosomal insertion of a transposon encoding both phoB and phoR, while chromosomal insertion of a transposon encoding phoB only partially regained alginate production and normal growth. Lack of PhoB will lead to decreased phosphate uptake under phosphate-limiting conditions, and this may result in less trinucleotides [23]. Furthermore, in Pseudomonas aeruginosa the AlgQ (AlgR2), has been shown to regulate the production of GTP through its positive regulatory effect on transcription of ndk, and Ndk is required for alginate production [24]. AlgQ is an anti-sigma-70 factor and has been shown to positively regulate alginate production [25], possibly by increasing the amount of RNAP available for the alternative sigma-factor AlgU. Transcription of algQ is positively regulated by PhoB [24]. In our strain, transcription of the alginate biosynthetic genes depends on the Pm promoter, which in turn depends on the sigma factors RpoH and RpoS for transcription [26]. Thus, it is possible that AlgQ may have a positive effect on expression from Pm. If that is the case, this might also explain the lack of alginate production in the phoB mutant when grown in a low phosphate medium.

Inactivation of certain genes involved in cell wall metabolism and vitamin biosynthesis leads to decreased alginate yield

In the present screen, insertions in five of the nine genes known to be involved in peptidoglycan recycling in Pseudomonas [27] were identified as having a negative impact on alginate biosynthesis (mpl, ampG, anmK, amgK and nagZ). The absence of Mpl, which is involved in recycling of the peptide part of peptidoglycan, only slightly decreased the alginate production. However, absence of any of the other four identified enzymes, AmpG, AnmK, AmgK or NagZ, resulted in very low alginate production in the PIA medium and reduced alginate yield in the DEF4 media (Table 1). The sugar phosphates used for peptidoglycan synthesis either originates from peptidoglycan recycling or is synthesized from Fru6P (Fig. 1b). Since Fru6P is also a precursor for alginate, depletion of this phosphorylated sugar would be expected to cause decreased alginate yield [2]. The nagZ and anmK genes were cloned on transposons, and shown to complement the deficiency in alginate production in the corresponding insertion mutants (Table 2).

Three of the identified genes, aceE1, ilvD and ispA were linked to pyruvate metabolism (Fig. 1b). aceE1 encodes a component of pyruvate dehydrogenase, which is an essential part of the central carbon metabolism. The viability of this mutant might be explained by the presence of other genes encoding AceE-like proteins in P. fluorescens. However, the aceE1 mutant grew more slowly than strain MS2, and hardly produced any alginate. ilvD encodes a dihydroxy-acid dehydratase that participates in the biosynthesis of branched amino acids and in the biosynthesis of pantothenate (vitamin B5) and coenzyme A. The ilvD mutant displayed a similar phenotype as the aceE1 strain in all three media (Table 2). The ispA mutant would be expected to have defects in the biosynthesis of isoprenoids, which would affect the biosynthesis of ubiquinone and the cell membrane. This mutant produced very low amounts of alginate when grown in PIA, while the phenotypes in the DEF4 media were more similar to the control strain (Table 2). All three mutants were complemented when the corresponding wild type genes were expressed from transposons (Table 2). Disruption of a pathway may often result in an increased flow to the immediate precursor for the missing enzyme, since the cell will perceive a lack of the end product. In the ispA and ilvD mutants this would lead to consumption of pyruvate, which then would have to be replenished by increasing the flow through the Entner-Doudoroff pathway (Fig. 1b). Pantothenate (needed for CoA) and ubiquinone are necessary for the anabolism and energy production of the cell, and the medium-dependent defects in growth and alginate yield displayed by the mutants might be caused by a lower content of these vitamins in peptone (PIA) compared to yeast extract (DEF4).

Deficiencies in purine or tryptophan biosynthesis reduce alginate yield

Eleven of the mutants identified in the screen turned out to have insertions in genes needed for purine biosynthesis (purHFLKE and amn). GTP is required for alginate biosynthesis as a precursor for both GDP-mannuronic acid and the signal molecule c-di-GMP (Fig. 1b). Three of the identified purine biosynthesis mutants (purE, purH and purL) were retested in deep-well plate cultivations and grew poorly in all media (Table 2). The purH strain was complemented when wild-type purH was expressed from a transposon, while the purE mutant was not complemented by expressing purE. This might, however, result from a polar effect on the downstream purK gene. Addition of adenine and thiamine to the media increased both growth and alginate yield for all three mutants (Table 2), strongly suggesting that the observed phenotypes were caused by deficiencies in the purine synthesis pathway.

In eight of the sequenced mutants, the transposon had disrupted genes putatively involved in amino acid biosynthesis (Table 1). Three of these, trpDEF, were genes involved in tryptophan synthesis. The mutants with insertions in trpD and trpF were investigated further and both could be complemented by inserting an intact corresponding gene on a transposon (Table 2). Furthermore, addition of tryptophan to the growth medium restored normal growth and alginate yield in both mutants (Table 2).

Both tryptophan and purine synthesis are linked to Fru6P through the pentose phosphate pathway (Fig. 1b). Defects in these biosynthetic pathways might affect alginate synthesis negatively by increasing the need for phosphoribosyl pyrophosphate (PRPP), and thus increase the flow from Fru6P to this intermediate. Since GTP is necessary for alginate biosynthesis, the observed phenotypes might also be caused by an insufficient supply of purines. Our results are corroborated by other studies demonstrating that de novo synthesis of purine is necessary for biofilm formation in P. fluorescens [28], and that tryptophan is important for biofilm formation in Salmonella enterica [29].

Disruption of several genes encoding proteins involved in protein folding and modification result in reduced alginate yield

Prc is a protease known to affect alginate biosynthesis in some mucA mutants of P. aeruginosa, and has been proposed to indirectly participate in alginate biosynthetic gene activation through MucA cleavage induced by cell wall stress [30, 31]. However, in our strain both algC and the alg operon are controlled by the Pm promoter, not by the endogenous AlgU-MucA-regulated promoters. Still, four independent prc mutants were identified as displaying a reduced alginate yield (Table 1). Our results therefore show that in P. fluorescens a prc mutation negatively affects alginate biosynthesis even in a mucA + strain. In addition the screen identified another peptidase belonging to the same family, SohB, which also negatively affected alginate yield when inactivated. This phenotype was complemented by a transposon expressing sohB (Table 2). It is unknown which proteins, apart from MucA, is the target of these two proteases in P. fluorescens.

Two genes encoding proteins involved in protein folding were identified in the screen as producing less alginate than the control (Table 1). PFLU4383 encodes a putative thiol:disulfide interchange protein and is located upstream of and partly overlapping dsbG, encoding another disulfide isomerase. Three independent inactivations of PFLU4383 were identified. PFLU5007 encodes the disulfide isomerase DsbC and its phenotype was complemented by a transposon-encoded copy of the gene (Table 2). A mutant of P. aeruginosa with transposon-inactivated dsbC was recently found to display a non-mucoid phenotype [32], indicating that DsbC is needed for normal levels of alginate production in both species. The results suggest that full alginate production in these media depend on correct folding of some proteins. It remains unknown which proteins need these isomerases for correct folding.

Conclusion

In an earlier study, it was shown that inactivation of glucose-6-phosphate dehydrogenase increased alginate yield when glycerol was used as carbon source, and this indicated that the availability of Fru6P may be one limiting factor to sustain high level alginate production [2]. Furthermore, it has been shown that the number of alginate biosynthetic complexes are not influenced by the absence of precursors for alginate synthesis [4], indicating that these complexes are not destabilized in the absence of polymer synthesis. The aim of screening a transposon insertion library, was to discover genes and metabolic pathways that indirectly influence alginate production in P. fluorescens. The main conclusion of our data is that alginate biosynthesis depends on sufficient levels of Fru6P, GTP and c-di-GMP (Fig. 1b). Inactivation of genes in several systems sensing the carbon/nitrogen ratio resulted in mutants that produce less alginate than the parent strain, and this further indicates that alginate production might be down-regulated as a response to a perceived carbon limitation. A majority of the analysed mutants displayed a significantly decreased alginate yield, while the cell yield was less affected, and in some cases even increased. This suggests that when P. fluorescens is facing certain nutrient limitations, less alginate is produced.

Methods

Growth of bacteria

E. coli and P. fluorescens (Table 4) were routinely cultivated in L broth or on L agar at 37 °C or 30 °C, respectively [33]. P. fluorescens was also grown in PIA medium [33], DEF4 medium [34] and DEF3 medium with low phosphate: KH2PO4 0,14 mg/L, KCl 0.36 g/L, NH4Cl 2.21 g/L, citric acid · H2O 0.9 g/L, ferric citrate 0.02 g/L, H3BO3 0.001 g/L, MnCl2 · 4H2O 0.005 g/L, EDTA · 2H2O 0.0039 g/L, CuCl2 · 2H2O 0.0005 g/L, Na2Mo4O4 · 2H2O 0.0008 g/L, CoCl2 · 6H2O 0.0008 g/L, Zn (CH3COO)2 · 2H2O 0.0027 g/L, NaCl 1.56 g/L, MgSO4 · 7H2O 0.57 g/L, MOPS 10 g/L. For precultures, 0.39 g/L yeast extract was added to the DEF4 medium. The pH of DEF3 and DEF4 was adjusted to 7.0. Carbon sources – fructose or glycerol – were added to 20 g/L. Antibiotics used: ampicillin (Ap, 200 mg/L), tetracycline (Tc, 15 mg/L), apramycin (Am, 25 mg/L), kanamycin (Km, 50 mg/L). For growth in microtiter plates and micro bioreactors (BioLector®), half the concentrations of the media containing 7 g/L carbon source was used, and the cultures were incubated at 25 °C as detailed previously [34]. For some experiments adenine (0.8 mM), thiamine (0.05 mM), or tryptophan (2.5 mM) were added as medium supplements. For growth studies in Biolector® microreactors the cultivations were performed in M2P-labs FlowerPlate® BOH with 1 ml medium per reactor. The cultivations were started (3 vol-% inoculum) from L broth precultures cultivated at 30 °C for 18 h. The BOH plates were incubated at 25 °C, 1300 rpm with 3 mm orbital movement at 80% humidity. pH, dissolved oxygen and biomass were measured automatically every hour by the Biolector system. The biomass measured by the Biolectors Photomultiplier was calibrated by offline optical density measurements using a standard spectrophotometer.

Table 4 Bacterial strainsa and plasmids used in this study

Analyses of alginate and growth

The cultures were incubated for three to four days before the cell density and alginate yield were assayed. Enzymatic measurements of alginate production were performed as described earlier [2, 35]. Briefly, the cell free medium were treated with a mixture of an M-specific and a G-specific alginate lyase, and OD230 before and after the reaction were measured using a Beckman Coulter robotic liquid handling work station with a Paradigm microplate reader.

Construction of the transposon vector and the transposon insertion library

Cloning, transformation, conjugation and gene deletions were performed as described earlier [33]. The plasmids and transposons used and constructed in this study are described in Table 4, while the primer sequences are found in Additional file 2: Table S1. PCR was performed using the Expand High Fidelity kit (Roche). PCR-amplified genes were confirmed by sequencing. Transposon insertions were to be identified by sequencing, so a transposon vector that would allow easy cloning of the transposon insertion site in E. coli was constructed and designated pMS11 (Table 4, Fig. 2c). The vector contains a derivative of the Tn5 minitransposon that comprises oriR6K and a gene encoding kanamycin resistance within the transposon boundaries. The transposon contains single sites for the restriction enzymes SacI and EcoRI close to the ends of the transposon. pMS11 was propagated in E. coli S17-1 λpir that encodes the Pir protein necessary for R6K-replication. pMS11 was transferred to P. fluorescens by conjugation, and conjugants were selected on PIA containing kanamycin. Colonies were picked using a Genetix QPixII colony picking robot and transferred to 384 well plates with 0.5 x PIA and Km, and incubated at 25 °C overnight before glycerol was added to 15% v/v and the plates were stored at −80 °C.

Identification of transposon insertion sites

Genomic DNA was isolated from mutants of interest. For some mutants the transposon insertion site was identified by direct sequencing using this DNA as the template and the primer MS11 Ori (Additional Table S1). For sequencing on genomic DNA, 5 μg DNA, 50 pmol sequencing primer, 8 μl 2.5x BigDye Terminator Ready Reaction Mix v1.1 (Applied Biosystems) and water to 20 μl was mixed. The reaction was subjected to sixty cycles of 30 s denaturation at 95 °C, 30 s annealing at 52 °C, and four minutes elongation at 60 °C. Alternatively, the DNA flanking the transposon insertion site was cloned by restricting genomic DNA isolated from a transposon mutant with SacI or EcoRI. The fragments were circularized by ligation, and the ligation mixture was transformed into E. coli S17-1 λpir and selected for resistance to kanamycin. Sequencing the resulting plasmids provided better quality sequences than by sequencing directly on genomic DNA. The transposon insertion points were identified by comparing the obtained sequences to the genome sequence (GenBank Accession number AM181176).

References

  1. 1.

    Andersen T, Strand BL, Formo K, Alsberg E, Christensen BE, et al. Alginates as biomaterials in tissue engineering. In: Rauter AP, Lindhorst TK, editors. Carbohydr Chem, 37. Cambridge, UK: The Royal Society of Chemistry; 2012. p. 227–58.

    Google Scholar 

  2. 2.

    Maleki S, Mærk M, Valla S, Ertesvåg H. Mutational analyses of glucose dehydrogenase and glucose-6-phosphate dehydrogenase genes in Pseudomonas fluorescens reveal their effects on growth and alginate production. Appl Environ Microbiol. 2015;81(10):3349–56.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Damron F, Goldberg J. Proteolytic regulation of alginate overproduction in Pseudomonas aeruginosa. Mol Microbiol. 2012;84(4):595–607.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Maleki S, Almaas E, Zotchev SB, Valla S, Ertesvåg H. Alginate biosynthesis factories in Pseudomonas fluorescens: localization and correlation with alginate production level. Appl Environ Microbiol. 2016;82(4):2027.2036.

    Article  Google Scholar 

  5. 5.

    Bakkevig K, Sletta H, Gimmestad M, Aune R, Ertesvåg H, Degnes K, Christensen BE, Ellingsen TE, Valla S. Role of the Pseudomonas fluorescens alginate lyase (AlgL) in clearing the periplasm of alginates not exported to the extracellular environment. J Bacteriol. 2005;187(24):8375–84.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Whitney JC, Whitfield GB, Marmont LS, Yip P, Neculai AM, Lobsanov YD, Robinson H, Ohman DE, Howell PL. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa. J Biol Chem. 2015;290(20):12451–62.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Hay ID, Remminghorst U, Rehm BH. MucR, a novel membrane-associated regulator of alginate biosynthesis in Pseudomonas aeruginosa. Appl Environ Microbiol. 2009;75(4):1110–20.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Withers TR, Yin Y, Yu HD. Identification of novel genes associated with alginate production in Pseudomonas aeruginosa using mini-himar1 mariner transposon-mediated mutagenesis. J Vis Exp. 2014;85.

  9. 9.

    Ramos JL, Marques S, Timmis KN. Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. Annu Rev Microbiol. 1997;51:341–73.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Gimmestad M, Sletta H, Karunakaran P, Bakkevig K, Ertesvåg H, Ellingsen TE, Skjåk-Bræk G, Valla S. New mutant strains of Pseudomonas fluorescens and variants thereof, methods of their production, and uses thereof in alginate production. In: WO2004/011628. 2002.

    Google Scholar 

  11. 11.

    Nishijyo T, Haas D, Itoh Y. The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa. Mol Microbiol. 2001;40(4):917–31.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Zhang XX, Rainey PB. Dual involvement of CbrAB and NtrBC in the regulation of histidine utilization in Pseudomonas fluorescens SBW25. Genetics. 2008;178(1):185–95.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Sonnleitner E, Valentini M, Wenner N, Haichar FZ, Haas D, Lapouge K. Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa. PLoS One. 2012;7(10):e44637.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Amador CI, Canosa I, Govantes F, Santero E. Lack of CbrB in Pseudomonas putida affects not only amino acids metabolism but also different stress responses and biofilm development. Environ Microbiol. 2010;12(6):1748–61.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Hervás AB, Canosa I, Little R, Dixon R, Santero E. NtrC-dependent regulatory network for nitrogen assimilation in Pseudomonas putida. J Bacteriol. 2009;191(19):6123–35.

  16. 16.

    Rehm N, Buchinger S, Strösser J, Dotzauer A, Walter B, Hans S, Bathe B, Schomburg D, Krämer R, Burkovski A. Impact of adenylyltransferase GlnE on nitrogen starvation response in Corynebacterium glutamicum. J Biotechnol. 2010;145(3):244–52.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Lee CR, Park YH, Kim M, Kim YR, Park S, Peterkofsky A, Seok YJ. Reciprocal regulation of the autophosphorylation of enzyme INtr by glutamine and alpha-ketoglutarate in Escherichia coli. Mol Microbiol. 2013;88(3):473–85.

  18. 18.

    Pflüger-Grau K, de Lorenzo V. From the phosphoenolpyruvate phosphotransferase system to selfish metabolism: a story retraced in Pseudomonas putida. FEMS Microbiol Lett. 2014;356(2):144–53.

    Article  PubMed  Google Scholar 

  19. 19.

    Lessie TG, Phibbs Jr PV. Alternative pathways of carbohydrate utilization in pseudomonads. Annu Rev Microbiol. 1984;38:359–88.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Velazquez F, Pfluger K, Cases I, De Eugenio LI, de Lorenzo V. The phosphotransferase system formed by PtsP, PtsO, and PtsN proteins controls production of polyhydroxyalkanoates in Pseudomonas putida. J Bacteriol. 2007;189(12):4529–33.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Cabeen MT, Leiman SA, Losick R. Colony-morphology screening uncovers a role for the Pseudomonas aeruginosa nitrogen-related phosphotransferase system in biofilm formation. Mol Microbiol. 2016;99(3):557–70.

  22. 22.

    Lamarche MG, Wanner BL, Crépin S, Harel J. The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis. FEMS Microbiol Rev. 2008;32(3):461–73.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Marzan LW, Shimizu K. Metabolic regulation of Escherichia coli and its phoB and phoR genes knockout mutants under phosphate and nitrogen limitations as well as at acidic condition. Microb Cell Fact. 2011;10:39.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Kim HY, Schlictman D, Shankar S, Xie Z, Chakrabarty AM, Kornberg A. Alginate, inorganic polyphosphate, GTP and ppGpp synthesis co-regulated in Pseudomonas aeruginosa: implications for stationary phase survival and synthesis of RNA/DNA precursors. Mol Microbiol. 1998;27(4):717–25.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Dove SL, Hochschild A. Bacterial two-hybrid analysis of interactions between region 4 of the σ70 subunit of RNA polymerase and the transcriptional regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa. J Bacteriol. 2001;183(21):6413–21.

  26. 26.

    Marqués S, Manzanera M, González-Pérez MM, Gallegos MT, Ramos J. The XylS-dependent Pm promoter is transcribed in vivo by RNA polymerase with σ32 or σ38 depending on the growth phase. Mol Microbiol. 1999;31(4):1105–13.

  27. 27.

    Gisin J, Schneider A, Nagele B, Borisova M, Mayer C. A cell wall recycling shortcut that bypasses peptidoglycan de novo biosynthesis. Nat Chem Biol. 2013;9(8):491–3.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Yoshioka S, Newell PD. Disruption of de novo purine biosynthesis in Pseudomonas fluorescens Pf0-1 leads to reduced biofilm formation and a reduction in cell size of surface-attached but not planktonic cells. PeerJ. 2016;4:e1543.

    Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Hamilton S, Bongaerts RJ, Mulholland F, Cochrane B, Porter J, Lucchini S, Lappin-Scott HM, Hinton JC. The transcriptional programme of Salmonella enterica serovar Typhimurium reveals a key role for tryptophan metabolism in biofilms. BMC Genomics. 2009;10:599.

    Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Wood LF, Leech AJ, Ohman DE. Cell wall-inhibitory antibiotics activate the alginate biosynthesis operon in Pseudomonas aeruginosa: Roles of sigma (AlgT) and the AlgW and Prc proteases. Mol Microbiol. 2006;62(2):412–26.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Reiling SA, Jansen JA, Henley BJ, Singh S, Chattin C, Chandler M, Rowen DW. Prc protease promotes mucoidy in mucA mutants of Pseudomonas aeruginosa. Microbiology. 2005;151(Pt 7):2251–61.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Damron FH, Barbier M, McKenney ES, Schurr MJ, Goldberg JB. Genes required for and effects of alginate overproduction induced by growth of Pseudomonas aeruginosa on Pseudomonas isolation agar supplemented with ammonium metavanadate. J Bacteriol. 2013;195(18):4020–36.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Gimmestad M, Sletta H, Ertesvåg H, Bakkevig K, Jain S, Suh S-j, Skjåk-Bræk G, Ellingsen TE, Ohman DE, Valla S. The Pseudomonas fluorescens AlgG protein, but not its mannuronan C5-epimerase activity, is needed for alginate polymer formation. J Bacteriol. 2003;185(12):3515–23.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Correa E, Sletta H, Ellis DI, Hoel S, Ertesvåg H, Ellingsen TE, Valla S, Goodacre R. Rapid reagentless quantification of alginate biosynthesis in Pseudomonas fluorescens bacteria mutants using FT-IR spectroscopy coupled to multivariate partial least squares regression. Anal Bioanal Chem. 2012;403(9):2591–9.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Østgaard K. Enzymatic microassay for the determination and characterization of alginates. Carbohydr Polym. 1992;19:51–9.

    Article  Google Scholar 

  36. 36.

    de Lorenzo V, Cases I, Herrero M, Timmis KN. Early and late response of TOL promoters to pathway inducers: Identification of postexponential promoters in Pseudomonas putida with lacZ-tet bicistronic reporters. J Bacteriol. 1993;175:6902–7.

    Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Rainey PB, Bailey MJ. Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome. Mol Microbiol. 1996;19(3):521–33.

    CAS  Article  PubMed  Google Scholar 

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Acknowledgments

The authors thank Elin Finstuen, Randi Aune and Sunniva Hoel for valuable technical assistance and Mali Mærk for helpful comments and discussions.

Funding

This work was supported by the Era-Net SYSMO project SCARAB, by the Norwegian Research Council (project 1459451), and by a strategic project at SINTEF.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and in the Additional file 2: Table S1 and Additional file 1: Figure S1.

Authors’ contributions

HE supervised the strain and library construction and annotated the mutants to functions. MS constructed the strain, transposon vector and library. HS and GK designed, developed and validated the screening protocols used for analyses and verification of mutant phenotypes. MS, GK and HS participated in the transposon screen, YQS identified inactivated genes and complemented some mutants, TK identified and complemented the phoBR mutants. HE, SV, HS and TE participated in the initiation and design of the study and in the writing of the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

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Not applicable.

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Correspondence to Helga Ertesvåg.

Additional information

Ertesvåg and Sletta contributed equally, and Senneset and Sun contributed equally.

Additional files

Additional file 1: Figure S1.

Growth profiles of Pseudomonas fluorescence SBW25 and MS2 cultivated in 0.5 x PIA. (PPTX 75 kb)

Additional file 2: Table S1.

Primers used in the study. (XLS 30 kb)

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Ertesvåg, H., Sletta, H., Senneset, M. et al. Identification of genes affecting alginate biosynthesis in Pseudomonas fluorescens by screening a transposon insertion library. BMC Genomics 18, 11 (2017). https://doi.org/10.1186/s12864-016-3467-7

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Keywords

  • Pseudomonas fluorescens
  • Alginate biosynthesis
  • Transposon mutants
  • Fructose 6-phosphate
  • Purine
  • Tryptophan
  • Peptidoglycan recycling