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. 2015 Apr;197(7):1276-87.
doi: 10.1128/JB.02408-14. Epub 2015 Feb 2.

The enhancer binding protein Nla6 regulates developmental genes that are important for Myxococcus xanthus sporulation

Krista M Giglio  1 , Chengjun Zhu  1 , Courtney Klunder  1 , Shelley Kummer  1 , Anthony G Garza  2
Affiliations

The enhancer binding protein Nla6 regulates developmental genes that are important for Myxococcus xanthus sporulation

Krista M Giglio et al. J Bacteriol. 2015 Apr.

Abstract

In the bacterium Myxococcus xanthus, starvation triggers the formation of multicellular fruiting bodies containing thousands of stress-resistant spores. Recent work showed that fruiting body development is regulated by a cascade of transcriptional activators called enhancer binding proteins (EBPs). The EBP Nla6 is a key component of this cascade; it regulates the promoters of other EBP genes, including a downstream-functioning EBP gene that is crucial for sporulation. In recent expression studies, hundreds of Nla6-dependent genes were identified, suggesting that the EBP gene targets of Nla6 may be part of a much larger regulon. The goal of this study was to identify and characterize genes that belong to the Nla6 regulon. Accordingly, a direct repeat [consensus, C(C/A)ACGNNGNC] binding site for Nla6 was identified using in vitro and in vivo mutational analyses, and the sequence was subsequently used to find 40 potential developmental promoter (88 gene) targets. We showed that Nla6 binds to the promoter region of four new targets (asgE, exo, MXAN2688, and MXAN3259) in vitro and that Nla6 is important for their normal expression in vivo. Phenotypic studies indicate that all of the experimentally confirmed targets of Nla6 are primarily involved in sporulation. These targets include genes involved in transcriptional regulation, cell-cell signal production, and spore differentiation and maturation. Although sporulation occurs late in development, all of the developmental loci analyzed here show an Nla6-dependent burst in expression soon after starvation is induced. This finding suggests that Nla6 starts preparing cells for sporulation very early in the developmental process.

Importance: Bacterial development yields a remarkable array of complex multicellular forms. One such form, which is commonly found in nature, is a surface-associated aggregate of cells known as a biofilm. Mature biofilms are structurally complex and contain cells that are highly resistant to antibacterial agents. When starving, the model bacterium Myxococcus xanthus forms a biofilm containing a thin mat of cells and multicellular structures that house a highly resistant cell type called a myxospore. Here, we identify the promoter binding site of the transcriptional activator Nla6, identify genes in the Nla6 regulon, and show that several of the genes in the Nla6 regulon are important for production of stress-resistant spores in starvation-induced M. xanthus biofilms.

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Figures

FIG 1
FIG 1
EMSAs performed with the Nla6 DBD and fragments of the asgEveg promoter. The binding reactions were performed with (+) or without (−) a 1 μM concentration of purified Nla6 DBD and an end-labeled fragment of the dev promoter region (negative control) or the end-labeled P1 or P2 fragment of the asgEveg promoter region.
FIG 2
FIG 2
Relative expression of asgE, actB, and nla28 in wild-type and nla6 mutant cells. Expression of asgE mRNA, actB mRNA, and nla28 mRNA in wild-type and nla6 mutant cells was determined using qPCR. (A) Growth of wild-type cells (squares) and nla6 mutant cells (circles). Cells were grown at 32°C in CTTYE broth and harvested at densities of 40, 150, and 250 Klett units. (B) asgE mRNA levels in wild-type (gray bars) and nla6 mutant (black bars) cells harvested during growth. Total cellular RNA was isolated from three independent biological replicates, cDNA was generated from each RNA sample, qPCRs were performed in triplicate, and the relative levels of asgE mRNA were calculated using the reference gene rpoD and the ΔΔCT method. Values are means. (C and D) actB (C) and nla28 (D) mRNA levels in cells harvested at 0, 1, 8, 12, and 24 h of development. Total cellular RNA from seven independent biological replicates was pooled, cDNA was generated from the pooled RNA, qPCRs were performed in triplicate, and the relative levels of actB mRNA and nla28 mRNA, respectively, were calculated by using the ΔΔCT method, with 16S rRNA as the reference. Values (C and D) are means and standard deviations of the means for wild-type cells (squares) and nla6 mutant cells (circles). The error bars in the actB and nla28 mRNA expression profiles do not extend past the symbols.
FIG 3
FIG 3
EMSAs performed with the Nla6 DBD and nla promoter fragments containing Nla6 enhancer element mutations. (A) nla28 promoter region. The 151-bp nla28 P1a fragment contains the wild-type Nla6 enhancer element nla28 EE1. The nla28 P1b and nla28 P1c fragments are derivatives of nla28 P1a that contain a CC-to-TT substitution in the first nla28 EE1 half-site and a CA-to-TT substitution in the second nla28 EE1 half-site, respectively. The mutated nucleotides in nla28 EE1 are in bold and underlined. (B) EMSAs performed with (+) or without (−) the Nla6 DBD and the nla28 P1a, nla28 P1b, or nla28 P1c promoter fragment. (C) nla6 promoter region. The nla6 P1a fragment contains the wild-type Nla6 enhancer element nla6 EE1. The nla6 P1b and nla6 P1c fragments are derivatives of nla6 P1a that contain a CAA-to-TTT substitution in the first nla6 EE1 half-site and a CCA-to-TTT substitution in the second nla6 EE1 half-site, respectively. The mutated nucleotides in nla6 EE1 are in bold and underlined. (D) EMSAs performed with (+) or without (−) the Nla6 DBD and the nla6 P1a, nla6 P1b, or nla6 P1c promoter fragment.
FIG 4
FIG 4
In vivo activity of the nla28 promoter after the introduction of Nla6 enhancer element mutations. (A) The 418-bp nla28 P3a fragment of the nla28 promoter region carries a wild-type σ54 promoter and an intact Nla6 enhancer element (nla28 EE1). nla28 P3b and nla28 P3c are derivatives of nla28 P3a that carry a mutation in the first and second nla28 EE1 half-site, respectively. The mutated nucleotides in nla28 EE1 are in bold and underlined. (B) nla28 P3a, nla28 P3b, and nla28 P3c were cloned into a lacZ expression vector and transferred to the wild-type M. xanthus strain DK1622. At various times during development, β-galactosidase-specific activities (defined as nanomoles of ONP produced per minute per milligram of protein) in cells carrying a wild-type or a mutant promoter fragment were determined. The values are mean β-galactosidase specific activities from 3 samples taken at 1 h of development, which is the time at which nla28 expression peaks. Error bars represent standard deviations of the means.
FIG 5
FIG 5
EMSAs performed with the Nla6 DBD and fragments of putative Nla6 target promoters identified using the consensus Nla6 enhancer. The binding reactions were performed with (+) or without (−) a 1 μM concentration of purified Nla6 DBD and the end-labeled negative-control dev promoter fragment, the exo P1 promoter fragment, the exo P2 promoter fragment, the MXAN2688 P2 promoter fragment, or the MXAN3259 P1 promoter fragment.
FIG 6
FIG 6
Developmental expression of exoA, MXAN3259, and MXAN2688 in wild-type and nla6 mutant cells. Expression of exoA mRNA, MXAN3259 mRNA, and MXAN2688 mRNA in wild-type and nla6 mutant cells was determined using qPCR. Mean exo mRNA levels (A), MXAN3259 mRNA levels (B) and MXAN2688 mRNA levels (C) in cells harvested at 0, 1, 2, 8, 12 and 24 h of development are shown. Total cellular RNA from seven independent biological replicates was pooled, cDNA was generated from the pooled RNA, qPCRs were performed in triplicate, and the relative levels of exo mRNA, MXAN3259 mRNA, and MXAN2688 mRNA, respectively, were calculated by the ΔΔCT method with 16S rRNA as the reference. Values are means for wild-type cells (squares) and nla6 mutant cells (circles). Error bars show standard deviations of the means. With one exception, the error bars in the exo, MXAN3259, and MXAN2688 mRNA expression profiles do not extend past the symbols.
FIG 7
FIG 7
Nla6-mediated regulation of sporulation in M. xanthus. The Nla6 targets analyzed here are primarily involved in sporulation. Each of these Nla6 targets can be placed into one of three functional categories: A-signal accumulation (asgE), transcriptional regulation (actB and nla28), or spore differentiation and maturation (exo, MXAN2688, and MXAN3259). We suggest that Nla6 directly activates (solid arrows) expression of the actB, exo, MXAN2688, MXAN3259, and nla28 loci early in development and indirectly (dashed arrows) inhibits or activates their expression late in development. We also suggest that Nla6 directly (solid arrow) activates asgE expression around the time that cells begin the transition into stationary-phase growth. In addition to confirmed Nla6 targets, 17 operons and 20 single genes were classified as potential targets. Many of the putative targets of Nla6 were classified as having regulatory, cell wall/membrane biogenesis, or solute transport functions. For a more detailed description of Nla6 and its targets and putative targets, see Discussion.
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