BMC Genomics

, 18:11 | Cite as

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

  • Helga Ertesvåg
  • Håvard Sletta
  • Mona Senneset
  • Yi-Qian Sun
  • Geir Klinkenberg
  • Therese Aursand Konradsen
  • Trond E. Ellingsen
  • Svein Valla
Open Access
Research article
Part of the following topical collections:
  1. Prokaryote microbial genomics

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.

Keywords

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

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

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

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

Number of independent transposon mutants

Gene ID

Gene

Function

Growth (G) and Alginate production (A) in different mediaa

    

PIA Gly

0.5xDEF4 Gly

0.5xDEF4 Fru

    

G

A

G

A

G

A

    

%

SD

%

SD

%

SD

%

SD

%

SD

%

SD

 

SBW25 WT

  

132

6

0

25

86

0

0

0

350

6

1

6

 

Control

  

100

6

100

13

100

2

100

13

100

5

100

3

 1

PFLU0460

aceE1

Energy production and conversion

66

2

63

2

57

4

19

4

293

20

0

4

 1

PFLU3193

aceE2

108

4

48

2

98

6

89

9

130

3

106

19

 1

PFLU5345

cioB

151

18

69

3

102

1

65

5

129

7

119

13

 2

PFLU3801

ftsK

Cell cycle control, cell division, chromosome partitioning

38

0

17

5

72

14

4

3

425

42

3

3

 

PFLU3801

ftsK

53

7

39

8

102

2

86

8

16

1

7

4

 1

PFLU5304

 

107

6

47

7

100

6

96

8

130

18

101

12

 1

PFLU1384

 

Amino acid transport and metabolism

108

3

18

3

71

1

81

33

148

45

95

6

 1

PFLU2019

 

126

27

0

3

116

14

0

1

338

14

0

0

 1

PFLU2124

 

127

4

40

7

102

2

88

7

294

30

55

27

 1

PFLU3475

 

106

2

45

11

98

7

88

9

132

5

99

32

 1

PFLU3887

 

104

4

12

11

93

6

72

16

132

28

53

5

 1

PFLU5797

ilvD

69

22

64

2

60

10

17

1

338

31

2

3

 1

PFLU4188

trpF

100

2

34

3

105

5

0

0

343

3

0

5

 1

PFLU5559

trpD

62

5

58

11

106

9

9

12

344

23

0

1

 2

PFLU5561

trpE

102

2

55

4

140

1

0

3

4

14

1

2

 1

PFLU0612

purH

Nucleotide transport and metabolism

44

5

4

2

81

3

0

0

317

13

0

0

 1

PFLU4183

purF

55

2

48

21

81

2

9

6

344

11

4

2

 4

PFLU5034

purL

50

1

43

10

70

1

1

3

348

7

0

2

 2

PFLU6054

purK

84

4

2

5

113

8

0

4

343

3

1

4

 1

PFLU6055

purE

88

7

45

25

87

5

37

5

368

9

0

2

 2

PFLU5396

amn

79

2

29

17

106

2

66

9

135

16

62

18

 2

PFLU1142

glpK

Carbohydrate transport and metabolism

52

2

66

39

19

7

2

4

214

9

52

40

 3

PFLU1143

glpF

59

2

4

2

18

3

0

2

128

10

80

4

 1

PFLU3030

paaF

109

5

33

7

94

4

84

18

122

7

118

16

 2

PFLU3365

treZ

107

2

62

12

97

1

98

3

132

17

132

4

 3

PFLU4630

acnA

144

8

73

14

92

7

68

23

125

3

113

20

 1

PFLU4949

pykA

107

4

41

14

51

3

132

5

160

3

85

12

 1

PFLU0416

hemE

Coenzyme transport and metabolism

147

5

68

8

98

2

66

14

117

11

136

12

 1

PFLU5820

nudH

Translation, ribosomal structure and biogenesis

77

3

23

12

78

0

127

5

65

23

31

3

 1

 

23S rRNA

152

10

52

16

98

1

94

14

130

15

142

24

 1

PFLU3173

 

Transcription

92

8

10

3

93

2

89

4

276

0

67

7

 1

PFLU3307

 

165

18

74

11

103

2

88

28

137

10

110

8

 1

PFLU4259

 

125

2

62

1

75

5

84

11

153

20

111

5

 1

PFLU4774

 

102

10

7

3

91

12

75

7

110

3

79

26

 1

PFLU5984

dut

Replication, recombination and repair

114

6

45

6

47

3

62

7

149

5

86

1

 1

PFLU0013

htrB

Cell wall/ membrane/ envelope biogenesis

117

7

40

5

78

5

62

3

123

24

110

18

 1

PFLU1562

nagZ

67

1

5

2

89

2

60

20

186

9

80

7

 1

PFLU4993

ampG

58

2

4

2

87

1

53

10

198

6

83

17

 1

PFLU5439

mpl

129

6

36

5

103

1

80

8

106

4

46

6

 2

PFLU5545

 

62

3

8

2

99

9

90

11

204

1

65

6

 2

PFLU5546

anmK

54

4

3

2

86

4

68

11

192

1

59

26

 3

PFLU5573

amgK

95

30

45

10

23

2

15

4

192

18

46

38

 1

PFLU5461

ispA

88

5

2

6

85

4

46

8

282

31

15

7

 1

PFLU4418

fleN

Cell motility

88

1

43

4

80

4

104

29

80

1

40

12

 1

PFLU4439

fliF

140

8

12

2

97

3

0

0

342

3

0

0

 1

PFLU4448

fliC

111

8

38

2

87

5

102

7

171

3

132

6

 1

PFLU0870

tldD

Posttransla-tional modification, protein turnover, chaperones

111

3

26

5

103

1

88

6

137

6

111

16

 4

PFLU2032

prc

100

2

37

6

81

3

59

6

124

7

26

6

 2

PFLU2614

sohB

98

3

11

3

96

1

73

8

108

7

109

12

 1

PFLU3805

clpA

147

7

60

7

104

1

127

20

256

8

124

21

 3

PFLU4383

 

118

6

21

8

87

22

51

5

298

8

38

13

 1

PFLU5007

dsbC

102

7

13

6

97

2

79

5

120

10

84

2

 2

PFLU5911

ppx

Inorganic ion transport and metabolism

50

2

82

38

73

3

67

28

90

13

76

2

 

PFLU5911

ppx

54

1

0

0

73

4

101

7

107

5

98

15

 1

PFLU0511

rsgA

General function prediction only

108

5

86

11

96

4

101

32

104

12

124

26

 1

PFLU2104

 

149

9

66

18

94

1

65

13

117

7

136

15

 1

PFLU2996

 

134

10

66

7

104

3

74

12

122

9

132

5

 1

PFLU3202

 

108

3

76

10

81

3

47

6

403

7

22

3

 1

PFLU3391

 

51

25

58

8

68

1

95

7

118

0

129

13

 1

PFLU3411

 

105

6

50

9

101

2

70

29

135

2

137

19

 1

PFLU3456

 

104

4

47

5

103

0

107

2

151

13

117

6

 1

PFLU1883

 

Function unknown

69

0

0

0

74

14

66

14

712

268

0

0

 1

PFLU1995

 

149

7

85

9

104

1

97

7

158

18

113

17

 1

PFLU4517

 

129

3

82

6

96

3

28

5

377

18

2

6

 1

PFLU5579

apaG

39

2

47

21

104

5

4

3

175

32

18

7

 1

PFLU2489

 

93

10

120

51

69

3

75

8

324

40

23

9

 1

PFLU5377

 

107

8

8

3

97

4

76

5

145

1

92

20

 1

Upstream PFLU2629

 

107

6

9

3

106

4

84

13

300

16

106

7

 1

Upstream PFLU3162

 

122

2

77

20

76

1

88

12

280

36

96

15

 1

Upstream PFLU3931

 

97

2

35

3

75

2

3

3

327

11

12

5

 1

PFLU2519

 

Pseudogene

114

4

148

26

102

3

98

6

337

9

89

13

 1

PFLU0259

ompR

Signal transduction mechanisms

89

5

7

1

101

4

70

12

127

17

101

12

 2

PFLU0461

glnE

85

14

10

4

120

4

104

13

317

11

67

1

 1

PFLU4125A

 

119

4

164

14

112

0

86

4

158

5

72

2

 4

PFLU5236

cbrA

140

1

34

7

105

4

55

7

319

5

66

21

 2

PFLU5237

cbrB

118

1

15

3

109

3

23

3

483

24

31

2

 2

PFLU5819

ptsP

80

12

8

3

90

6

79

15

49

1

5

5

 1

PFLU6039

phoB

78

1

44

20

102

2

90

16

110

7

84

21

 1

PFLU6040

phoR

25

0

0

0

97

12

87

0

99

22

100

23

 1

PFLU2808

 

100

2

11

3

110

4

75

5

255

52

19

11

 1

PFLU3002

 

Intracellular trafficking, secretion, and vesicular transport

153

6

48

15

93

5

83

6

141

9

94

5

 1

PFLU3951

 

106

6

8

1

105

2

83

1

110

5

101

20

 1

PFLU5567

 

98

9

61

5

0

0

0

0

59

1

2

4

a: The strains were cultivated in microtiter plates for three days before cell and alginate yield were measured. The mutants shown are those that displayed significantly different alginate production levels in at least one of the three tested media. Data are not shown for strains with transposon insertions in the genes encoded by the alginate operon or in algC. The Table shows how many independent transposon insertions mutants that were identified for each gene, the gene identifier, the gene name, and which functional group the corresponding protein is assigned to. Growth above 125% and alginate production above 110% are marked using bold types, growth and alginate production between 10 and 50% are marked using italics, and growth and alginate production below 10% are written in bold italics. Three biological replicates were cultivated for each strain, and the results are given as percent (%) of the values obtained from the control strain MS2. Standard deviations for the three replicates are shown in the columns to the right (SD)

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

Inactivated gene

Supplement/comple-menting gene (s)b

PIA

0.5xDEF4 Glycerol

0.5xDEF4 Fructose

Growthc

Alginate

Growthc

Alginate

Growth

Alginate

wt

 

100

100

100

100

100

100

trpF

 

65

0

145

0

291

38

tryptophane

70

139

78

60

130

45

trpF

88

121

88

94

153

106

trpD

 

56

0

1

0

3

0

tryptophane

68

261

68

80

71

75

trpD

85

142

114

23

242

38

trpDC

89

85

81

91

152

120

purH

 

29

0

1

0

21

0

Adenine, thiamine

51

188

14

33

39

21

purH

90

100

91

88

163

97

purE

 

33

0

0

0

1

0

Adenine, thiamine

56

91

17

50

43

29

purE

57

0

1

0

1

0

purL

 

19

0

0

0

1

0

Adenine, thiamine

52

124

12

48

38

30

ilvD

 

77

18

2

2

4

0

ilvD

91

82

91

109

222

97

aceEI

 

33

58

6

20

13

7

aceEI

96

42

85

96

244

123

PFLU3030

 

88

0

80

89

104

103

PFLU3030

97

142

89

123

101

111

dsbC

 

108

52

111

89

149

115

dsbC

105

127

105

117

94

92

sohB

 

100

9

100

50

479

−2

sohB

91

109

104

106

109

100

nagZ

 

53

0

139

93

115

75

nagZ

87

145

84

120

289

79

anmK

 

32

0

124

98

197

99

anmK

84

118

86

135

111

128

ispA

 

82

0

100

97

121

115

ispA

92

212

88

117

405

77

cbrB

 

83

0

110

63

374

46

cbrB

91

118

91

95

213

107

PFLU3887

 

91

67

98

103

105

110

PFLU3887

90

33

109

103

112

103

PFLU5567

 

87

64

2

6

141

30

PFLU5567

93

103

1

16

6

10

a: The strains were grown in deep-well plates containing the indicated media for four days before cell and alginate yield were measured. b: empty field denotes no supplement or complementing vector. c: Values are given as percentage of the control strain (SBW25 MS1 ΔalgC:: TnKB61). Actual values for the control strain were (growth [OD660]/alginate [g/L]): PIA: 0.492/0.33, DEF4 glycerol: 0.850/1.72, DEF4 fructose: 0.308/3.08. Growth above 125% and alginate production above 110% are marked using bold types, growth and alginate production between 10 and 50% are marked using italics, and growth and alginate production below 10% are written in bold italics

Table 3

Effect of PhoBR disruptions on P. fluorescens growth and alginate biosynthesis

Strain

Growth (OD600)

Alginate (g/l)

SBW25mucAHE230

2.5+/−0.24

4.3+/−0.89

SBW25mucAHE230 ΔphoR

2.7+/−0.33

3.6+/−0.36

SBW25mucAHE230 ΔphoB

1.4+/−0.12

0.0+/−0.0

SBW25mucAHE230 ΔphoR:: TnTK5

2.4+/−0.09

4.0+/−0.24

SBW25mucAHE230 ΔphoR:: TnTK7

2.1+/−0.10

4.3+/−0.66

SBW25mucAHE230 ΔphoB:: TnTK6

1.5+/−0.20

1.2+/−0.56

SBW25mucAHE230 ΔphoB:: TnTK7

2.0+/−0.13

5.5+/−0.12

a: The cells were grown for 72 h in shaking flasks using DEF3 medium with 20 g/l glycerol, 1 μM phosphate and 0.5 mM m-toluate. Average values from three independent experiments are shown

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

Strains

Description

Reference

E. coli S17-1 (λpir)

λpir (for replication of oriR6K-plasmids) recA, thi pro hsdR-M + RP4 2-Tc::Mu-Km::Tn7TpRSMR

[36]

P. fluorescens SBW25

Non-mucoid P. fluorescens wild type

[37]

SBW25MS1

Derivative of SBW25 where the Pm promoter is inserted directly upstream of algD using pMS9.

This study

SBW25MS1 ΔalgC:: TnKB61

Derivative of SBW25 MS1 where algC has been deleted utilizing pKB22, and a copy of algC controlled by the PmG5 promoter has been introduced on a transposon inserted into gene PFLU2944.

This study

SBW25mucAHE230

Alginate-producing derivative of SBW25 encoding a defect MucA and where the expression of algC is controlled by the PmG5 promoter

H. Ertesvåg, unpublished

SBW25mucAHE230ΔphoB

Derivative of SBW25mucAHE230 where an in-frame deletion in phoB was introduced utilizing pTK10.

This study

SBW25mucAHE230ΔphoR

Derivative of SBW25mucAHE230 where an in-frame deletion in phoR was introduced utilizing pTK9.

This study

Plasmids

  

pKD20

pUT based transposon vector containing PmG5. Apr, Kmr.

[5]

pLitmus28Tc

High copy number cloning vector. Tcr, Apr

[5]

pMG48

RK2-based gene replacement vector. lacZ + , Tcr, Apr

[33]

pMC1

RK2-based gene replacement vector for replacing the DNA sequence upstream of algD with the Pm-promoter. lacZ + , Tcr, Apr

[10]

pKB22

Gene replacement vector for creating an algC-deletion. lacZ + , Tcr, Apr

[5]

pKB60

Transposon vector. Contains the transposon TnKB60 with algC under the control of PmG5. Apr, Kmr

[5]

pYQ1

pUT based transposon vector containing PmG5. Amr, Kmr.

[2]

pEM1

Derivative of pLitmus28Tc containing part of the transposon from pKD20. Apr

[2]

pKB61

Derivative of pKB60 where a 1.7 kb AvrII-NcoI DNA fragment encoding Kmr and most of XylS was exchanged with a 2.5 kb AvrII-NotI DNA fragment containing tetAR. Tcr, Apr

This study

pMS9

Derivative of pMC1 where a 0.7 kb SbfI-NotI DNA fragment containing a gene upstream of algD was exchanged with a PCR product containing the 0.8 kb sequence directly upstream of algD. lacZ + , Tcr, Apr

This study

pMS2

Derivative of pLitmus28Tc where the tetAR genes were exchanged with a 3.4 kb BamHI-fragment from pKD20 containing the minitransposon and oriR6K. Kmr, Apr

This study

pMS10

Derivative of pMS2 where a 0.5 kb BsiWI-EcoRI-fragment containing oriR6K was deleted and the 1.9 kb NotI-PstI fragment encoding XylS was exchanged with a 0.4 kb PCR product encoding oriR6K. Kmr, Apr

This study

pMS11

Derivative of pKD20 where a 3.7 kb BssHII-SfiI-fragment was exchanged with a 1.5 kb BssHII-SfiI-fragment containing oriR6K from pMS10. Kmr, Apr

This study

pTK1

Derivative of pEM1 in which a 2.2 kb PCR-amplified NdeI-NotI DNA fragment encoding phoBR from P. fluorescens was inserted. Apr.

This study

pTK3

Derivative of pTK1 in which an inserted 2.2 kb PCR-amplified NcoI-PspOMI DNA fragment from P. fluorescens including the first 46 nt of phoB replaced most of the phoB gene. Apr.

This study

pTK4

Derivative of pTK1 from which a 0.9 kb BstEII-BsaBI DNA fragment encoding most of phoR was deleted. Apr.

This study

pTK5

Derivative of pKD20 in which a 1.5 kb NdeI-NotI PCR fragment from P. fluorescens containing phoR was inserted. Kmr.

This study

pTK6

Derivative of pKD20 in which a 1.1 kb NcoI-NotI DNA fragment from pTK4 containing phoB was inserted. Kmr.

This study

pTK7

Derivative of pKD20 in which a 2.2 kb NdeI-NotI PCR fragment from P. fluorescens containing phoBR was inserted. Kmr.

This study

pTK8

Derivative of pTK1 from which a 0.3 kb BstEII DNA fragment was deleted, creating an in-frame deletion in phoR. Apr.

This study

pTK9

Derivative of pMG48 in which a 3.0 kb NcoI-NotI DNA fragment from pTK8 was inserted, containing a deletion in phoR. Apr, Tcr.

This study

pTK10

Derivative of pMG48 in which a 3.1 kb NcoI-NotI DNA fragment from pTK3 was inserted, containing a deletion in phoB. Apr, Tcr.

This study

pYQ1 trpF

Derivative of pYQ1 in which a 0.7 kb NdeI-NotI PCR fragment encoding TrpF was inserted. Amr.

This study

pYQ1 trpD

Derivative of pYQ1 in which a 1.1 kb NdeI-NotI PCR fragment encoding TrpD was inserted. Amr.

This study

pYQ1 trpDC

Derivative of pYQ1 in which a 1.9 kb NdeI-NotI PCR fragment encoding TrpDC was inserted. Amr.

This study

pYQ1 purH

Derivative of pYQ1 in which a 1.6 kb NdeI-NotI PCR fragment encoding PurH was inserted. Amr.

This study

pYQ1 purE

Derivative of pYQ1 in which a 0.5 kb NdeI-NotI PCR fragment encoding PurE was inserted. Amr.

This study

pYQ1 ilvD

Derivative of pYQ1 in which a 2.1 kb NdeI-NotI PCR fragment encoding IlvD was inserted. Amr.

This study

pYQ1 aceEI

Derivative of pYQ1 in which a 2.7 kb NdeI-NotI PCR fragment encoding AceE1 was inserted. Amr.

This study

pYQ1 PFLU3030

Derivative of pYQ1 in which a 1.0 kb NdeI-NotI PCR fragment encoding PFLU3030 was inserted. Amr.

This study

pYQ1 PFLU3887

Derivative of pYQ1 in which a 1.0 kb NdeI-NotI PCR fragment encoding PFLU3887 was inserted. Amr.

This study

pYQ1 PFLU5567

Derivative of pYQ1 in which a 1.2 kb NdeI-NotI PCR fragment encoding PFLU5567 was inserted. Amr.

This study

pYQ1 dsbC

Derivative of pYQ1 in which a 0.9 kb NdeI-NotI PCR fragment encoding DsbC was inserted. Amr.

This study

pYQ1 sohB

Derivative of pYQ1 in which a 1.1 kb NdeI-NotI PCR fragment encoding SohB was inserted. Amr.

This study

pYQ1 nagZ

Derivative of pYQ1 in which a 1.1 kb NdeI-NotI PCR fragment encoding NagZ was inserted. Amr.

This study

pYQ1 anmK

Derivative of pYQ1 in which a 1.5 kb NdeI-NotI PCR fragment encoding AnmK was inserted. Amr.

This study

pYQ1 ispA

Derivative of pYQ1 in which a 0.9 kb NdeI-NotI PCR fragment encoding IspA was inserted. Amr.

This study

pYQ1 cbrB

Derivative of pYQ1 in which a 1.4 kb NdeI-NotI PCR fragment encoding CbrB was inserted. Amr.

This study

a: Mutant strains complemented with transposons are not included in the Table

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).

Notes

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.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Supplementary material

12864_2016_3467_MOESM1_ESM.pptx (76 kb)
Additional file 1: Figure S1. Growth profiles of Pseudomonas fluorescence SBW25 and MS2 cultivated in 0.5 x PIA. (PPTX 75 kb)
12864_2016_3467_MOESM2_ESM.xls (33 kb)
Additional file 2: Table S1. Primers used in the study. (XLS 30 kb)

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Copyright information

© The Author(s). 2017

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.Department of BiotechnologyNTNU-Norwegian University of Science and TechnologyTrondheimNorway
  2. 2.SINTEF Materials and ChemistryTrondheimNorway

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