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. 2012 Sep;194(17):4698-708.
doi: 10.1128/JB.00225-12. Epub 2012 Jun 29.

The Nla28S/Nla28 two-component signal transduction system regulates sporulation in Myxococcus xanthus

Zaara Sarwar  1 , Anthony G Garza
Affiliations

The Nla28S/Nla28 two-component signal transduction system regulates sporulation in Myxococcus xanthus

Zaara Sarwar et al. J Bacteriol. 2012 Sep.

Abstract

The response regulator Nla28 is a key component in a cascade of transcriptional activators that modulates expression of many important developmental genes in Myxococcus xanthus. In this study, we identified and characterized Nla28S, a histidine kinase that modulates the activity of this important regulator of M. xanthus developmental genes. We show that the putative cytoplasmic domain of Nla28S has the in vitro biochemical properties of a histidine kinase protein: it hydrolyzes ATP and undergoes an ATP-dependent autophosphorylation that is acid labile and base stable. We also show that the putative cytoplasmic domain of Nla28S transfers a phosphoryl group to Nla28 in vitro, that the phosphotransfer is specific, and that a substitution in the predicted site of Nla28 phosphorylation (aspartate 53) abolishes the phosphotransfer reaction. In phenotypic studies, we found that a mutation in nla28S produces a developmental phenotype similar to, but weaker than, that produced by a mutation in nla28; both mutations primarily affect sporulation. Together, these data indicate that Nla28S is the in vivo histidine kinase partner of Nla28 and that the primary function of the Nla28S/Nla28 two-component signal transduction system is to regulate sporulation genes. The results of genetic studies suggest that phosphorylation of Nla28S is important for the in vivo sporulation function of the Nla28S/Nla28 two-component system. In addition, the quorum signal known as A-signal is important for full developmental expression of the nla28S-nla28 operon, suggesting that quorum signaling regulates the availability of the Nla28S/Nla28 signal transduction circuit in developing cells.

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Figures

Fig 1
Fig 1
Organization of the nla28S-nla28 operon, domain organization of relevant proteins, and an Nla28S sequence alignment. (A) Organization of the nla28S-nla28 operon. (B) Domain organization of Nla28S (476 amino acids), GST-Nla28S-cyt (an N-terminal GST fusion to the C-terminal 270 amino acids of Nla28S), Nla28 (447 amino acids), and GST-Nla28 (an N-terminal GST fusion to full-length Nla28). (C) Alignment of the Nla28S amino acid sequence with those of other well-characterized HKs indicates that Nla28S has all of the conserved HK sequence motifs. The alignment was generated using ClustalW2. The H-, N-, D-, F-, and G-boxes are shown, and the conserved residues are in black and bold type. HKs from Escherichia coli (Ec), Pseudomonas fluorescens (Pf), Shigella flexneri (Sf), and Myxococcus xanthus (Mx) are shown. GST, glutathione S-transferase; DHp, dimerization and histidine phosphorylation domain; CA, catalytic and ATPase domain; REC, receiver domain; AAA, ATPase domain; HTH, helix-turn-helix domain.
Fig 2
Fig 2
ATP hydrolysis activity of GST-Nla28S. A standard colorimetric assay that couples the hydrolysis of ATP to the oxidation of NADH was used to determine ATP hydrolysis activity of GST-Nla28S-cyt. The plot shows the concentration of ATP hydrolyzed by GST-Nla28S-cyt versus the time of the reaction. The initial ATP concentrations in the reactions were 0.1 mM (filled circles), 0.3 mM (open triangles), and 2 mM (open squares). Each measurement was done in triplicate. Error bars represent standard errors of the means of three replicates.
Fig 3
Fig 3
In vitro autophosphorylation activity of GST-Nla28S-cyt. (A) Time course of the autophosphorylation activity of GST-Nla28S-cyt (GST-Nla28S-cyt∼P) incubated with [γ-32P]ATP at room temperature. (B) Autophosphorylation activity of GST-Nla28S-cyt incubated with [γ-32P]ATP for 60 min and treated with 0.1 N HCl or 1 N NaOH for 20 min at room temperature. (C) Autophosphorylation activity of GST-Nla28S-cyt, GST-Nla28S-cyt H242A, and GSTNla28S-cyt D386A incubated with [γ-32P]ATP for 60 min at room temperature.
Fig 4
Fig 4
In vitro phosphotransfer assays. (A) GST-Nla28 was incubated with [γ-32P]ATP or [32P]acetyl phosphate ([32P]AcPO4) for 30 min. (B) Autophosphorylated GST-Nla28S-cyt (GST-Nla28S-cyt∼32P) was incubated with GST-Nla28 at room temperature. (C) Autophosphorylated GST-Nla28S-cyt was incubated with His-OmpR or in kinase buffer alone at room temperature for 60 min. (D) Autophosphorylated His-EnvZ-cyt was incubated in kinase buffer alone, with GST-Nla28, or with His-OmpR at room temperature for 60 min. (E) Autophosphorylated GST-Nla28S-cyt was incubated in kinase buffer alone, with GST-Nla28, or with GST-Nla28 D53A at room temperature for 60 min.
Fig 5
Fig 5
Development phenotypes of wild-type (WT) and Δnla28S mutant cells. Wild-type and Δnla28S cells were spotted onto TPM agar plates (top two panels) or CF agar plates (bottom two panels), and the progress of fruiting-body development was monitored for 5 days using a Nikon Eclipse model E400 microscope at a total magnification of ×40. Photographs were taken at 0, 24, 48, 72, and 120 h poststarvation.
Fig 6
Fig 6
Expression of nla28S in wild-type and asgB mutant cells. (A) qPCR was used to examine developmental expression of nla28S in wild-type (DK1622) cells. The nla28S expression levels shown are relative to the levels found in growing wild-type cells (0 h). The values are means derived from three replicates. The error bars indicate standard deviations of the means. (B) qPCR was used to examine developmental expression of nla28S in asgB mutant (DK4398) cells at the time of its peak expression (2 h poststarvation) in wild-type cells. The nla28S expression levels shown are relative to the levels found in wild-type cells at 2 h poststarvation. The values are means derived from three replicates. The error bars are standard errors of the means.
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