Protein kinases play central roles in the regulation of eukaryotic and prokaryotic cell growth, division, and differentiation. The Caulobacter crescentus divL gene encodes a novel bacterial tyrosine kinase essential for cell viability and division. Although the DivL protein is homologous to the ubiquitous bacterial histidine protein kinases (HPKs), it differs from previously studied members of this protein kinase family in that it contains a tyrosine residue (Tyr-550) in the conserved H-box instead of a histidine residue, which is the expected site of autophosphorylation. DivL is autophosphorylated on Tyr-550 in vitro, and this tyrosine residue is essential for cell viability and regulation of the cell division cycle. Purified DivL also catalyzes phosphorylation of CtrA and activates transcription in vitro of the cell cycle-regulated fliF promoter. Suppressor mutations in ctrA bypass the conditional cell division phenotype of cold-sensitive divL mutants, providing genetic evidence that DivL function in cell cycle and developmental regulation is mediated, at least in part, by the global response regulator CtrA. DivL is the only reported HPK homologue whose function has been shown to require autophosphorylation on a tyrosine, and, thus, it represents a new class of kinases within this superfamily of protein kinases.

The transcription factor hXBP-1 belongs to the family of basic region/leucine zipper (bZIP) proteins and interacts with the cAMP responsive element (CRE) of the major histocompatibility complex (MHC) class II A alpha, DR alpha and DP beta genes. However, the developmental expression of hXBP-1 as revealed by in situ hybridization in mouse embryos, has suggested that it interacts with the promoter of additional genes. To identify other potential target genes of this factor, we performed binding site selection experiments with recombinant hXBP-1 protein. The results indicated that hXBP-1 binds preferably to the CRE-like element GAT-GACGTG(T/G)NNN(A/T)T, wherein the core sequence ACGT is highly conserved, and that it also binds to some TPA response elements (TRE). hXBP-1 can transactivate multimers of the target sequences to which it binds in COS cells, and the level of transactivation directly correlates with the extent of binding as observed in gel retardation experiments. One target sequence that is strongly bound by hXBP-1 is the 21 bp repeat in the HTLV-1 LTR, and we demonstrate here that hXBP-1 can transactivate the HTLV-1 LTR. Further, the transactivation domain of hXBP-1 encompasses a large C-terminal region of the protein, containing domains rich in glutamine, serine and threonine, and proline and glutamine residues, as shown in transient transfection experiments using hXBP-1-GAL4 fusion proteins and a reporter gene under the control of GAL4-binding sites.

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The periodic and sequential expression of flagellar (fla) genes in the Caulobacter crescentus cell cycle depends on their organization into levels I to IV of a regulatory hierarchy in which genes at the top of the hierarchy are expressed early in the cell cycle and are required for the later expression of genes below them. In these studies, we have examined the regulatory role of level IIfliF operon, which is located near the top of the hierarchy. The last gene in the fliF operon, flbD, encodes a transcriptional factor required for activation of j54-dependent promoters at levels III and IV and negative autoregulation of the level II fliF promoter. We have physically mapped the fliF operon, identified four new genes in the transcription unit, and determined that the organization of these genes is 5'-fliF-fliG-flbE-fliN-flbD-3'. Three of the genes encode homologs of the MS ring protein (FliF) and two switch proteins (FliG and FliN) of enteric bacteria, and the fourth encodes a predicted protein (FlbE) without obvious similarities to known bacterial proteins. We have introduced nonpolar mutations in each of the open reading frames and shown that all of the newly identified genes (fliFfliG,flbE, andfliN) are required in addition toflbD for activation of the u54-dependentflgK andflbG promoters at level III. In contrast,fliFfliG, andflbE, but notfliN, are required in addition toflbD for negative autoregulation of the level II fliF promoter. The simplest interpretation of these results is that the requirements of FlbD in transcriptional activation and repression are not identical, and we speculate that FlbD function is subject to dual or overlapping controls. We also discuss the requirement of multiple structural genes for regulation of levels II and III genes and suggest thatfla gene expression in C. crescentus may be coupled to two checkpoints in flagellum assembly.The procaryotic flagellum is a complex organelle composed of three structural elements: the basal body or motor, which is embedded in the membrane and peptidoglycan layers of the cell envelope; the external hook, which attaches the basal body to the flagellar filament; and the filament itself, which rotates to move the cell (35, 62). In the dimorphic bacterium Caulobacter crescentus, the flagellum is assembled at one pole of the dividing cell late in the cell cycle and then segregates with the motile swarmer cell at division. Its biosynthesis requires the activity of 40 to 50 flagellar (fla) genes (15), and, as in the enteric bacteria, expression of these genes is regulated by their organization in a regulatory hierarchy in which expression of genes at the top of the hierarchy is required for expression of genes lower in the hierarchy (reviewed in references 7 and 44). In the C. crescentus hierarchy, there is also evidence that fla gene interactions mediate the negative, as well as the positive, transcriptional control of gene expression (45,49,70).A unique feature of flagellum biosynthesis in C. crescentus is the cell cycle regulation of fla gene expre...

We have characterized flbF, a key locus located at the top of the flagellar gene hierarchy of Caulobacter crescentus. This gene is required for transcription from a54 promoters of fla genes expressed late in the cell cycle. We have determined the nucleotide sequence of the gene, mapped the 5' end of the flbF RNA, and examined the pattern of expression in the cell cycle. Our results show thatflbF is expressed earlier in the cell cycle than other fla genes, that it is expressed at a low level throughout the stalked cell cycle, and that its 5' regulatory region contains sequences that can be aligned with the r28 promoter consensus reported for enteric bacteria. flbF contains an open reading frame of 700 residues with an amino-terminal half rich in hydrophobic residues that could correspond to six to eight transmembrane domains. The translated flbF sequence is very similar to LcrD (low calcium response) encoded by virulence plasmids of pathogenic Yersinia spp. (G. Plano, S. Barve, and S. Straley, J. Bacteriol. 173:7293-7303, 1991). LcrD and FlbF can be aligned over the entire length of the proteins with the greatest degree of sequence identity (45%) in the hydrophobic amino-terminal region. The high degree of sequence homology of proteins derived from widely differing organisms, including Caulobacter and Yersinia species, suggests that FlbF and LcrD may be representatives of a larger family of regulatory proteins with a common sensor mechanism for modifying responses to appropriate stimuli.Caulobacter crescentus is an aquatic bacterium that forms two distinct morphological cell types at division: the stalked cell that enters the cell division pathway, assembles a flagellum at the pole opposite the stalk, and divides asymmetrically to produce the mother stalked cell, and a new, flagellated swarmer cell. The swarmer cell, which also has pili and phage receptors localized at the flagellated cell pole, must develop further to lose these structures and eventually form a stalk at the same pole before it initiates DNA replication (17). The stage-specific formation of the flagellum in the C. crescentus cell cycle results from the expression of factor that is a homolog of the E. coli NtrC protein (26) and is required for transcription offlaN (14) and flbG (21).We report here initial studies on flbF (transcription unit IV) which is at the highest level of the regulatory hierarchy and is required along with genes in the flaO operon for transcriptional activation of late fla genes (18