A cell-type-specific protein-protein interaction modulates transcriptional activity of a master regulator in Caulobacter crescentus.

Progression through the Caulobacter cell cycle is driven by the master regulator CtrA, an essential two-component signaling protein that regulates the expression of nearly 100 genes. CtrA is abundant throughout the cell cycle except immediately prior to DNA replication. However, the expression of CtrA-activated genes is generally restricted to S phase. We identify the conserved protein SciP (small CtrA inhibitory protein) and show that it accumulates during G1, where it inhibits CtrA from activating target genes. The depletion of SciP from G1 cells leads to the inappropriate induction of CtrA-activated genes and, consequently, a disruption of the cell cycle. Conversely, the ectopic synthesis of SciP is sufficient to inhibit CtrA-dependent transcription, also disrupting the cell cycle. SciP binds directly to CtrA without affecting stability or phosphorylation; instead, SciP likely prevents CtrA from recruiting RNA polymerase. CtrA is thus tightly regulated by a protein-protein interaction which is critical to cell-cycle progression.

Regulated proteolysis of a transcription factor complex is critical to cell cycle progression in Caulobacter crescentus.

Cell cycle transitions are often triggered by the proteolysis of key regulatory proteins. In Caulobacter crescentus, the G1-S transition involves the degradation of an essential DNA-binding response regulator, CtrA, by the ClpXP protease. Here, we show that another critical cell cycle regulator, SciP, is also degraded during the G1-S transition, but by the Lon protease. SciP is a small protein that binds directly to CtrA and prevents it from activating target genes during G1. We demonstrate that SciP must be degraded during the G1-S transition so that cells can properly activate CtrA-dependent genes following DNA replication initiation and the reaccumulation of CtrA. These results indicate that like CtrA, SciP levels are tightly regulated during the Caulobacter cell cycle. In addition, we show that formation of a complex between CtrA and SciP at target promoters protects both proteins from their respective proteases. Degradation of either protein thus helps trigger the destruction of the other, facilitating a cooperative disassembly of the complex. Collectively, our results indicate that ClpXP and Lon each degrade an important cell cycle regulator, helping to trigger the onset of S phase and prepare cells for the subsequent programmes of gene expression critical to polar morphogenesis and cell division.

The cis-regulatory system of the tbrain gene: Alternative use of multiple modules to promote skeletogenic expression in the sea urchin embryo.

The genomic cis-regulatory systems controlling regulatory gene expression usually include multiple modules. The regulatory output of such systems at any given time depends on which module is directing the function of the basal transcription apparatus, and ultimately on the transcription factor inputs into that module. Here we examine regulation of the Strongylocentrotus purpuratus tbrain gene, a required activator of the skeletogenic specification state in the lineage descendant from the embryo micromeres. Alternate cis-regulatory modules were found to convey skeletogenic expression in reporter constructs. To determine their relative developmental functions in context, we made use of recombineered BAC constructs containing a GFP reporter and of derivatives from which specific modules had been deleted. The outputs of the various constructs were observed spatially by GFP fluorescence and quantitatively over time by QPCR. In the context of the complete genomic locus, early skeletogenic expression is controlled by an intron enhancer plus a proximal region containing a HesC site as predicted from network analysis. From ingression onward, however, a dedicated distal module utilizing positive Ets1/2 inputs contributes to definitive expression in the skeletogenic mesenchyme. This module also mediates a newly discovered negative Erg input which excludes non-skeletogenic mesodermal expression.

Anaerobic carbon monoxide dehydrogenase diversity in the homoacetogenic hindgut microbial communities of lower termites and the wood roach.

Anaerobic carbon monoxide dehydrogenase (CODH) is a key enzyme in the Wood-Ljungdahl (acetyl-CoA) pathway for acetogenesis performed by homoacetogenic bacteria. Acetate generated by gut bacteria via the acetyl-CoA pathway provides considerable nutrition to wood-feeding dictyopteran insects making CODH important to the obligate mutualism occurring between termites and their hindgut microbiota. To investigate CODH diversity in insect gut communities, we developed the first degenerate primers designed to amplify cooS genes, which encode the catalytic (ß) subunit of anaerobic CODH enzyme complexes. These primers target over 68 million combinations of potential forward and reverse cooS primer-binding sequences. We used the primers to identify cooS genes in bacterial isolates from the hindgut of a phylogenetically lower termite and to sample cooS diversity present in a variety of insect hindgut microbial communities including those of three phylogenetically-lower termites, Zootermopsis nevadensis, Reticulitermes hesperus, and Incisitermes minor, a wood-feeding cockroach, Cryptocercus punctulatus, and an omnivorous cockroach, Periplaneta americana. In total, we sequenced and analyzed 151 different cooS genes. These genes encode proteins that group within one of three highly divergent CODH phylogenetic clades. Each insect gut community contained CODH variants from all three of these clades. The patterns of CODH diversity in these communities likely reflect differences in enzyme or physiological function, and suggest that a diversity of microbial species participate in homoacetogenesis in these communities.