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Keywords:

  • Myxococcus xanthus ;
  • bacterial tyrosine kinase;
  • exopolysaccharide;
  • yellow pigment

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Myxococcus xanthus BtkB is composed of an N-terminal periplasmic domain and a C-terminal cytoplasmic tyrosine kinase domain. The C-terminal cytoplasmic domain of BtkB was autophosphorylated in the presence of [γ-32P]ATP and MgCl2, and the autophosphorylated BtkB was detected with antiphosphotyrosine antibody, suggesting that BtkB is a bacterial tyrosine (BY) kinase. BY kinases have been demonstrated in the production of extracellular polysaccharide (EPS), antibiotic resistance, stress response, and DNA metabolism. Myxococcus xanthus btkB gene was expressed mainly in the growth phase and early stages of fruiting body development. When cultured in nutrient medium at high temperature (37 °C), btkB mutant showed reduced maximum cell density as compared to the wild type. Under starvation conditions, btkB mutant cells formed fruiting bodies and spores about 24 h later than the wild-type strain. The btkB mutant overproduced yellow pigment during development. Also, btkB mutant showed a decrease in EPS production when compared with the wild-type strain. These results suggested that BtkB may play multiple roles in M. xanthus cells.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Myxococcus xanthus is a Gram-negative soil bacterium that exhibits a complex life cycle and social behavior. This bacterium has two genetically distinct motility systems: adventurous (A) motility and social (S) motility (Hodgkin & Kaiser, 1979). The A-motility system allows movement of isolated cells and does not require cell–cell contact, while the S-motility system is typically employed for coordinated group movement of cells. The S-motility in M. xanthus involves the interaction between two organelles, type IV pili and exopolysaccharide (EPS). When deprived of nutrients, thousands of cells move by gliding toward centers of aggregation to multicellular fruiting bodies, where the long vegetative rods change to spherical optically refractile cells with resistance properties (Reichenbach, 1986).

Bacteria are able to adapt to a wide variety of environmental conditions through the regulation of gene expression, and they use sophisticated signal transduction mechanisms to control specific gene expression. In bacteria, protein phosphorylation is catalyzed mainly by histidine kinases, which are key enzymes of the so-called ‘two-component systems’ (Laub & Goulian, 2007). From recent genomic analysis, eukaryotic-like protein serine/threonine kinases were found in various bacteria and coexist with histidine kinases (Pereira et al., 2011). In addition to these protein kinases, the presence of bacterial tyrosine (BY) kinases has been proven in several bacterial species (Shi et al., 2010). BY kinases have been shown to be mainly involved in the production of capsular polysaccharide (CPS) and EPS (Cuthbertson et al., 2009). For example, in Escherichia coli, tyrosine kinases, Wzc and Etk, have been reported to participate in the synthesis and transportation of CPS (Whitfield, 2006). Also, BY kinases have been found to phosphorylate heat-shock sigma and antisigma factors and single-stranded DNA-binding proteins (Klein et al., 2003; Mijakovic et al., 2006), suggesting that BY kinases are also involved in the heat-shock response and DNA metabolism. They show no sequence similarity with eukaryotic protein kinases. BY kinases from Gram-negative bacteria have two functional domains, N-terminal periplasmic and C-terminal cytoplasmic domains encoded by a single gene (Doublet et al., 2002). By contrast, BY kinases from Gram-positive bacteria are usually separated into two distinct proteins.

Genome data analysis revealed the possibility that M. xanthus possesses two BY kinases, a Gram-negative type BY kinase (MXAN_1025) and a Gram-positive type BY kinase (BtkA: MXAN_3228). We previously reported that M. xanthus BtkA has phosphorylation activity in the presence of a receptor protein Exo (MXAN_3227; Kimura et al., 2011). Phosphorylated BtkA was expressed late after starvation induction and early after glycerol induction, and BtkA was required for the formation of mature spores. In this study, we investigated the functional role of a Gram-negative type of BY kinase, BtkB, in M. xanthus.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Growth and development

Myxococcus xanthus FB (IFO 13542) was grown in Casitone-yeast extract (CYE) medium (Campos et al., 1978). The maximum cell density was determined with a hemocytometer. To obtain fruiting bodies, vegetative cells were washed with 10 mM Tris–HCl, pH 7.5, and 8 mM MgSO4 (TM buffer) and spotted onto clone fruiting (CF) agar plates (Hagen et al., 1978).

Expression and purification of recombinant BtkB

The part of the btkB gene encoding a cytoplasmic domain was amplified by PCR using btkBEN and btkBEC primers (Supporting information, Table S1), and then the amplified 800-bp DNA fragment was cloned into an expression vector, pCold TF (Takara Bio). Protein expression in transformed E. coli was induced by incubation at 15 °C for 24 h and the addition of 0.1 mM isopropyl-β-d-1-thiogalactopyranoside. The cytoplasmic domain of BtkB was purified by affinity chromatography on a Talon CellThru column (Clontech).

Site-directed and deletion mutagenesis

The btkB gene cloned into the vector pCold TF was used as a template for PCR. Two site-directed mutations of tyrosine residues to phenylalanine residues and a C-terminal tyrosine cluster deletion mutation were generated by the PrimeSTAR mutagenesis basal kit (Takara Bio) using the primers (Table S1). The resulting PCR products were transformed into E. coli BL21 (DE3). After confirmation of the desired mutations by DNA sequencing, the mutant enzymes were expressed and purified by the methods described previously.

Construction of btkB mutant

The btkBMN and btkBMC primers were used to amplify the DNA fragment containing the btkB gene from the M. xanthus FB genome. The PCR product was ligated into a pBluescript SK vector (Toyobo), and then MscI fragments (total 1.4-kb) of the btkB gene were deleted. A kanamycin resistance gene (1.25-kb) was inserted into MscI sites of the btkB gene, and the resulting disrupted gene was amplified by PCR using the aforementioned primers. The PCR product thus obtained was introduced into M. xanthus FB cells by electroporation (Plamann et al., 1992). Myxococcus xanthus kanamycin-resistant colonies were grown in CYE medium containing kanamycin (100 μg mL−1), and chromosomal DNAs were prepared from the mutants. Using PCR and restriction enzyme analyses, we confirmed that the kanamycin resistance gene was inserted into the btkB gene on the chromosomes of M. xanthus mutant.

Kinase activity assay

The autophosphorylation assay was performed in 20 μL of 40 mM Tris–HCl buffer (pH 7.0), 1 mM DTT, 5 mM MgCl2, 5 μM ATP, and 0.15 MBq [γ-32P]ATP at 37 °C for 30 min. The reaction was loaded onto a 12.5% SDS-PAGE gel, which was autoradiographed and analyzed by BAS1800 (Fuji film). For Western blot analysis, the cytoplasmic domain of BtkB was incubated with 0.1 mM ATP, 1 mM DTT, and 5 mM MgCl2 at 37 °C for 1 h. Also, ATPase activity was performed in 20 μL of 40 mM Tris–HCl buffer (pH 7.0), 1 mM DTT, 5 mM MgCl2, 1 mM ATP, and 2 μg BtkB at 37 °C for 60 min. Released phosphate was measured with the malachite green reagent (Enzo life sciences).

Western blot analyses

Myxococcus xanthus wild-type and btkB mutant cells were grown in CYE medium and harvested in the exponential growth phase and stationary phase. Also, developmental cells were prepared on CF agar plates or CYE medium containing 0.5 M glycerol. Approximately 2 × 107 cells were dissolved in SDS sample buffer, and denatured proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were then transferred to PVDF membranes for Western blotting. The membranes were incubated with a horseradish peroxidase–conjugated antiphosphotyrosine PY20 (Santa Cruz Biotechnology). Blots were developed with ECL reagent (GE Healthcare).

Quantitative reverse transcriptase (qRT) PCR

Total RNA was isolated from exponential and stationary phase cells and during cell development at 24, 48, and 72 h and then treated with DNase (Promega). After inactivation of DNase, cDNA was synthesized from the RNA samples (each 0.8 μg) using PrimeScript II RTase (Takara Bio Inc.) and random hexamers, and PCR was performed with Kapa SYBR Fast qPCR master mix (KAPABiosystems), primers (RTbtkBN and RTbtkBC, Table S1), and the synthesized cDNA using the ABI 7300 real-time cycler. The mRNA levels of a downstream gene (MXAN_1029) were also determined by qRT-PCR analysis using the primers (RT1029N and RT1029C, Table S1). A control without reverse transcriptase was performed to detect residual contaminating genomic DNA.

Congo red and trypan blue binding assays

Exponential phase cells (8 × 108 cells mL−1) in CYE medium were used for the assays. Cells were harvested, washed, and resuspended at approximately 5 × 108 cells mL−1 in TM buffer. A total of 360 μL of the cell suspension was mixed with 40 μL dye stock solution (150 μg mL−1 Congo red and 100 μg mL−1 trypan blue). The assay was performed as previously described (Black & Yang, 2004).

Polysaccharide measurement

Cells grown in CYE medium were harvested in the exponential growth phase and stationary phase, washed three times with distilled water, and then sonicated without glass beads three times for 1 min each. Cells were also starved on CF agar and harvested at 48 and 96 h. The cells were sonicated with glass beads five times for 1 min each. The supernatant and pellet were separated by centrifugation three times at 10 000 g for 10 min. The pellet was washed three times with distilled water. The sugar contents of the supernatant and pellet suspension were determined at 490 nm by the phenol–sulfuric acid method, with glucose as the standard (Dubois et al., 1956).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Functional motifs of BtkB

BtkB consists of 710 amino acid residues with a calculated molecular mass of 78.4 kDa. The cytoplasmic domain of BtkB possesses a similar structural feature to Wzc homologues (Fig. 1) and shared homology with these BY kinases (29–32% identity). BtkB was an integral membrane protein harboring two transmembrane domains (amino acids 12–30 and 419–438) flanking a large periplasmic loop and had a cytoplasmic C-terminal region with a Walker A, A′, and B ATP-binding motif and a tyrosine-rich C terminus (Y-cluster). The phosphorylated form of the Y-cluster could be stabilized by interaction with a positively charged arginine- and lysine-rich flexible loop region (RK-cluster) of a neighboring subunit (Lee et al., 2008). The RK-cluster (amino acids 465–484) also existed in BtkB. The phosphorylation of ‘internal’ tyrosine residues, Y569 in Wzc and Y574 in Etk, is essential for Wzc and Etk kinase activities (Grangeasse et al., 2002; Lee et al., 2008). Also, BY kinase from Gram-negative bacteria contain a conserved arginine residue (R609 in Wzc and R614 in Etk) between Walker A and B motifs. The ‘internal’ tyrosine residues block the active site, and interaction of phosphorylated ‘internal’ tyrosine residue with arginine residue would unblock the catalytic site and, as a result, activate the kinase (Lee et al., 2008). However, BtkB does not possess a tyrosine or arginine residue in this position.

image

Figure 1. Alignment of the deduced cytoplasmic kinase domain of BtkB with the cytoplasmic domains of Wzc and Etk from Escherichia coli, the cytoplasmic domain of Ptk from Acinetobacter johnsonii, and the cytoplasmic domain of EpsB from Ralstonia solanacearum. Amino acid residues with agreement of more than three residues are indicated by filled boxes. The conserved RK-cluster, Walker motifs, and C-terminal Y-cluster are in boxes. Tyr-574 and Arg-614 in Etk are indicated by closed circles.

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In vitro autophosphorylation assay of BtkB

To determine whether BtkB has tyrosine kinase activity, recombinant BtkB protein was overexpressed and purified from E. coli; however, BtkB was not expressed in E. coli when the btkB gene was cloned into pCold TF and pCold vectors. It is reported that the periplasmic region of Wzc has no effect on the extent of phosphorylation of the C-domain (Grangeasse et al., 2002); therefore, a cytoplasmic fragment (Ser444-Ser710)-coding region of the btkB gene was amplified by PCR using primers and cloned into a pCold TF vector. The expression plasmid was transferred to E. coli BL21 (DE3). The fusion protein [trigger factor (TF; 52 kDa)-BtkB] with an N-terminal hexahistidine tag was expressed in the soluble fraction in E. coli. The fusion protein produced was purified by affinity chromatography, and then the purified BtkB was analyzed by SDS-PAGE, which revealed a single band corresponding to a molecular mass of 82 kDa (Fig. 2a). The value obtained by SDS-PAGE corresponded well with the molecular mass calculated from the predicted amino acid sequence of TF-tagged BtkB (81.0 kDa).

image

Figure 2. (a) SDS-PAGE analysis of purified cytoplasmic domain of BtkB. (b) Autophosphorylation of cytoplasmic domain of BtkB. BtkB was incubated with [γ-32P]ATP in the presence of various metal ions, and this reaction mixture was loaded onto SDS-PAGE. Phosphorylated protein was visualized by autoradiography. (c) Immunoblot using antiphosphotyrosine antibody. Cytoplasmic domain of BtkB was incubated in phosphorylation buffer (40 mM Tris–HCl, pH 7.0, 5 mM MgCl2, 1 mM DTT, and 0.1 mM ATP) for 1 h at 37 °C and incubated further with (+) or without (−) antarctic phosphatase for 30 min at 37 °C. (d) Autophosphorylation activity of BtkB mutants. After kinase reactions, tyrosine phosphorylation was measured by Western blot using PY20. Lane 1, BtkB; 2, BtkBY690F/Y693F mutant; 3, BtkBY686F/Y690F/Y693F/Y696F/Y699F mutant; 4, BtkB deletion mutant.

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The purified cytoplasmic domain of BtkB was incubated with [γ-32P] ATP in the presence of Mg2+, Mn2+, or Co2+ ion and analyzed by SDS-PAGE and autoradiography. As shown in Fig. 2b, autophosphorylation activity was only achieved in the presence of Mg2+ ion. Also, phosphorylation of BtkB was detected by Western immunoblotting with antiphosphotyrosine monoclonal antibody (PY20; Fig. 2c), indicating that BtkB is a tyrosine protein kinase. The cytoplasmic domain of Wzc from E. coli has been shown to harbor ATPase activity (Soulat et al., 2007), but the cytoplasmic domain of BtkB did not show ATPase activity (data not shown). As the mutation of Tyr 569 to Ala in Wzc led to a reduction in ATP hydrolysis activity, BtkB may not have ATPase activity. BtkB contains a Y-cluster, which contains five tyrosine residues. To determine whether cytoplasmic BtkB (Ser444-Ser710) autophosphorylates on tyrosine residues in the Y-cluster, a double mutant (Y690F/Y693F), a quintuple mutant (Y686F/Y690F/Y693F/Y696F/Y699F), and a deletion mutant lacking the Y-cluster (amino acids 686–699) were constructed. Mutant lacking two tyrosine residues (Y690 and Y693) was still autophosphorylated, although mutants lacking all tyrosine residues in the Y-cluster showed a great reduced level of autophosphorylation, suggesting that BtkB undergoes autophosphorylation on tyrosine residues in the Y-cluster (Fig. 2d).

Tyrosine-phosphorylated proteins in wild-type and btkB mutant cells

Changes in the tyrosine phosphorylation states in wild-type and btkB mutant cells during the growth phase and starvation- and glycerol-induced development were detected by SDS-PAGE and Western blotting using antiphosphotyrosine antibody (PY20; Fig. 3). In wild-type cells, a 79-kDa tyrosine-phosphorylated protein was mainly detected during growth phases and after 24 h of starvation-induced development and decreased during starvation- or glycerol-induced spore formation. The tyrosine-phosphorylated protein at 79 kDa was not expressed in btkB mutant cells. The value of 79 kDa corresponded well with the molecular mass (78.4 kDa) of BtkB. Tyrosine-phosphorylated protein at 26 kDa in btkB mutant cells appeared approximately 24 h later than in wild-type cells.

image

Figure 3. Expression of tyrosine phosphorylation proteins during development. (a) Starvation-induced development. Cells were grown in CYE medium and harvested in the exponential growth phase (E) and stationary phase (S). Also, cells were starved on CF agar and harvested at 24 h (24), 48 h (48), 72 h (72), or 96 h (96). (b) Glycerol-induced development. Wild-type and btkB mutant cells were incubated in CYE medium containing 0.5 M glycerol for 0, 1.5, 3.0, and 24 h. Harvested cells were boiled in SDS sample buffer, separated by SDS-12.5% PAGE, and analyzed by immunoblotting with PY20. The estimated molecular mass (kDa) of each protein is shown in brackets.

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qRT-PCR for analysis of expression of btkB gene

The timing and level of the expression of the btkB gene were also determined by qRT-PCR analysis. qRT-PCR analysis using cycle threshold values showed that the btkB gene was mainly expressed during the exponential phase and after 24 h of starvation-induced development. The expression levels of btkB gene gradually decreased during development. The relative cDNA quantities at 48 and 72 h of development were 66 ± 21% and 25 ± 6%, respectively, of that at 24 h (defined as 100 ± 18%). The btkB gene (MXAN_1025) forms an operon with four genes (MXAN_1026, 1027, 1028, and 1029). We also confirmed that MXAN_1029 gene, the last gene in the operon, in btkB mutant was transcribed at similar levels to wild type (113 ± 13% of wild-type level) in the exponential phase using qRT-PCR, suggesting that the phenotypes of the btkB mutant were not because of polar effects.

Phenotypes of btkB mutant

Growth and motility phenotypes

When btkB mutant cells were grown with shaking in CYE medium, wild-type and btkB mutant strains showed similar growth rates during exponential growth at optimal (28 °C) and high (37 °C) temperatures; however, compared with the wild-type strain, the maximum cell densities of the btkB mutant strain (2.9 × 109 cells mL−1) cultured at 28 °C were slightly decreased by about 15%, and when cultured at 37 °C, the btkB mutant strain further reduced the maximum cell density (3.2 × 108 cells mL−1) by roughly half (Fig. 4). Escherichia coli Etk has been found to be involved in heat-shock response by phosphorylating two heat-shock sigma factors, RpoH and RpoE (Klein et al., 2003). BtkB may also be involved in the heat-shock response. After reaching the maximum density, btkB mutant began to decrease rapidly. The cellular reversal of M. xanthus gliding is regulated by chemotaxis homologues (Shi et al., 2000). btkB mutant cells reversed direction approximately every 4.2 min on average, which was similar to that of wild-type cells (4.6 min).

image

Figure 4. Growth of wild-type (open circles) and btkB mutant (closed circles) cells at 28 °C (solid lines) and 37 °C (dashed lines). Growth was monitored by measuring optical density at 600 nm after vortex mixing. The experiments were performed in triplicate, and similar results were observed in every experiment.

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Developmental phenotype

The wild-type and btkB mutant strains (9 × 109 cells mL−1) were cultured on CF agar. The wild-type cells moved to aggregation centers and then formed mound-shaped fruiting bodies by 48–72 h on CF agar. After 3 days of development, the wild-type strain had produced dark fruiting bodies containing refractile sonication- and heat-resistant spores, while the btkB mutant strain had produced only translucent aggregates that were not filled with refractile spores (Fig. 5a). The btkB mutant cells formed fruiting bodies about 24 h later than the wild-type strain. The viable spore numbers produced by the mutant at 4 and 5 days were approximately 20–30% those of the wild-type strain; however, the final yield of viable spores for btkB mutant at 7 days was similar to that of the wild-type strain (Fig. 5b). Gram-positive type of M. xanthus BY kinase, BtkA, is required for the formation of mature spores (Kimura et al., 2011), while BtkB is not essential to form mature spores.

image

Figure 5. Fruiting body and spore formations of Myxococcus xanthus wild-type and btkB mutant strains. (a) Fruiting body development on CF agar. Photographs of fruiting bodies were taken after 3 days of incubation. (b) Numbers of spores form wild-type (open circles) and btkB mutant strains (closed circles) on CF agar. These strains (9 × 109 cells mL−1) were starved on CF agar at 28 °C for 7 days. After sonication for 1 min and incubation at 60 °C for 15 min, viable spores were determined by plating out serial dilutions on Casitone–Tris (CTT) agar plates. Data are expressed as the means of triplicate experiments. Standard deviations are shown by error bars.

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The btkB mutant produced a high level of yellow pigment during fruiting body formation (data not shown). The fruiting bodies of btkB mutant were harvested by gently scraping the surface with a bacteria spreader and were suspended in TM buffer. After centrifugation, the supernatant had a UV absorption maximum of 380 nm. This is in agreement with the major yellow pigment, DKxanthene-534, of M. xanthus DK1622 (λmax = 379 nm), which is essential for developmental sporulation (Meiser et al., 2006).

On the other hand, when vegetative cells were cultured with 0.5 M glycerol in CYE medium, the mutant cells sporulated at the same rate as wild-type cells (data not shown).

Examination of EPS production

EPS is an important component for social behaviors in M. xanthus, including gliding motility and fruiting body formation. EPS is the binding target for type IV pili in S-motility (Li et al., 2003) and forms a scaffold within the fruiting body structure (Shimkets, 1986; Lux et al., 2004). EPS production was analyzed quantitatively by the binding of Congo red and trypan blue. Exponentially growing cells (8 × 108 cells mL−1) in CYE medium were used for the assays, and the percentage of dye bound by cells was determined. btkB mutant cells bound Congo red and trypan blue at lower levels than wild-type cells. The btkB mutant bound 23.8 ± 0.2% and 7.1 ± 1.3% of Congo red and trypan blue, respectively, compared with 40.3 ± 0.1% and 29.8 ± 0.3% for the wild type.

We also determined the amount of EPS from broken cell pellets. As shown in Fig. 6, both growing cells and developing cells of btkB mutant contained a lower (61–79%) level of carbohydrate than wild-type cell pellets (defined as 100%). The reduction in EPS production in btkB mutant may cause a delay in the formation of fruiting bodies and spores. Different chemotaxis proteins and type IV pili of M. xanthus are required for EPS production (Yang et al., 2000; Bellenger et al., 2002). These data suggested that BtkB is not essential for, but plays a partial role in, the production of EPS.

image

Figure 6. EPS content of wild-type (open bars) and btkB mutant (closed bars) strains. Data are expressed as the means of triplicate experiments. Exponential (E) and stationary phase (S) cells and developing cells harvested from CF agar plates at 48 h (D48) and 96 h (D96) were sonicated, and then the pellet was separated by centrifugation. The sugar contents of the pellet suspension were determined by the phenol–sulfuric acid method.

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In this study, we showed the possibility that BtkB has multiple roles in M. xanthus cells. To understand the function of BtkB in M. xanthus, further work is needed to determine the substrates of BtkB in vivo.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (22570187).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
fml2651-sup-0001-TableS1.docWord document43KTable S1. Oligonucleotides used in this study.

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