Lucille Shapiro

Cell-cycle control of a cloned chromosomal origin of replication from Caulobacter crescentus

Caulobacter crescentus cell division is asymmetric and yields distinct swarmer cell and stalked cell progeny. Only the stalked cell initiates chromosomal replication, and the swarmer cell must differentiate into a stalked cell before chromosomal DNA replication can occur. In an effort to understand this developmental control of replication, we employed pulsed-field gel electrophoresis to localize and to isolate the chromosomal origin of replication. The C. crescentus homologues of several Escherichia coli genes are adjacent to the origin in the physical order hemE, origin, dnaA and dnaK,J. Deletion analysis reveals that the minimal sequence requirement for autonomous replication is greater than 430 base-pairs, but less than 720 base-pairs. A plasmid, whose replication relies only on DNA from the C. crescentus origin of replication, has a distinct temporal pattern of DNA synthesis that resembles that of the bona fide C. crescentus chromosome. This implies that cis-acting replication control elements are closely linked to this origin of replication. This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E. coli-like 13-mer, and an exceptional A + T-rich region. Point mutations in one of the DnaA boxes abolish replication in C. crescentus. This origin also possesses three additional motifs that are unique to the C. crescentus origin of replication: seven 8-mer (GGCCTTCC) motifs, nine 8-mer (AAGCCCGG) motifs, and five 9-mer (GTTAA-n7-TTAA) motifs are present. The latter two motifs are implicated in essential C. crescentus replication functions, because they are contained within specific deletions that abolish replication.

An unusual promoter controls cell-cycle regulation and dependence on DNA replication of the Caulobacter fliLM early flagellar operon

Transcription of flagellar genes in Caulobacter crescentus is programmed to occur during the predivisional stage of the cell cycle. The mechanism of activation of Class II flagellar genes, the highest identified genes in the Caulobacter flagellar hierarchy, is unknown. As a step toward understanding this process, we have defined cis‐acting sequences necessary for expression of a Class II flagellar operon, fliLM. Deletion analysis indicated that a 55 bp DNA fragment was sufficient for normal, temporally regulated promoter activity. Transcription from this promoter‐containing fragment was severely reduced when chromosomal DNA replication was inhibited. Extensive mutational analysis of the promoter region from ‐42 to ‐5 identified functionally important nucleotides at ‐36 and ‐35, between ‐29 and ‐22 and at ‐12, which correlates well with sequences conserved between fliLM and the analogous regions of two other Class II flagellar operons. The promoter sequence does not resemble that recognized by any known bacterial sigma factor. Models for regulation of Caulobacter early flagellar promoters are discussed in which RNA polymerase containing a novel Sigma subunit interacts with an activation factor bound to the central region of the promoter.

Expression of positional information during cell differentiation in caulobacter

The asymmetric targeting of proteins to the Caulobacter predivisional cell poles yields dissimilar progeny. We show that the products of transcriptional reporter gene fusions to a flagellin gene and to the flagellar hook operon are segregated to the progeny swarmer cell. This segregation does not depend on sequences within the mRNA, but on the upstream regulatory region. The subset of developmentally regulated flagellar genes that exhibit mRNA segregation has the same upstream cis-acting elements: an activator-binding site known as the ftr sequence and an IHF-binding site. We propose that these genes are preferentially transcribed from the chromosome in the incipient swarmer cell pole of the predivisional cell.

Plasmid and chromosomal DNA replication and partitioning during the Caulobacter crescentus cell cycle

Cell division in Caulobacter crescentus yields a swarmer and a stalked cell. Only the stalked cell progeny is able to replicate its chromosome, and the swarmer cell progeny must differentiate into a stalked cell before it too can replicate its chromosome. In an effort to understand the mechanisms that limit chromosomal replication to the stalked cell, plasmid DNA synthesis was analyzed during the developmental cell cycle of C. crescentus, and the partitioning of both the plasmids and the chromosomes to the progeny cells was examined. Unlike the chromosome, plasmids from the incompatibility groups Q and P replicated in all C. crescentus cell types. However, all plasmids tested showed a ten- to 20-fold higher replication rate in the stalked cells than the swarmer cells. We observed that all plasmids replicated during the C. crescentus cell cycle with comparable kinetics of DNA synthesis, even though we tested plasmids that encode very different known (and putative) replication proteins. We determined the plasmid copy number in both progeny cell types, and determined that plasmids partitioned equally to the stalked and swarmer cells. We also reexamined chromosome partitioning in a recombination-deficient strain of C. crescentus, and confirmed an earlier report that chromosomes partition to the progeny stalked and swarmer cells in a random manner that does not discriminate between old and new DNA strands.

Organization and ordered expression of Caulobacter genes encoding flagellar basal body rod and ring proteins

The biogenesis of the polar flagellum in Caulobacter crescentus is limited to a specific time in the cell cycle and to a specific site on the cell. The basal body is the first part of the flagellum to be assembled. In this report we identify a cluster of genes encoding basal body components and describe their transcriptional regulation. The genes in this cluster form an operon whose expression is controlled temporally. The first two genes encode homologs of FlgF and FlgG, which are the proximal and distal rod proteins, respectively. The sequences of the N and C termini of the Salmonella typhimurium flagellar axial proteins, rod, hook and HAP-1, known to be highly conserved, share a high degree of sequence identity with the FlgF and FlgG rod proteins of the distantly related, C. crescentus. Two additional genes in the flgF, flgG operon, flaD and flgH, both encode proteins with potentially cleavable signal sequences. The flgH gene, encoding the L-ring protein, is also transcribed from an internal promoter. Transcription from the flgF promoter initiates prior to initiation at the internal flgH promoter. The internal promoter and its activator site reside within the C-terminal coding sequence of the upstream flaD gene. This type of gene overlap is also observed in bacterial genes involved in cell division. Flagellum biogenesis, like cell division, is a morphogenic event that requires the orderly assembly of component proteins and the overlapping gene organization may affect this ordering of assembly. The promoters for the flgF operon and the flgH gene use sigma 54 to initiate transcription. The use of sigma 54 promoters, known to require cognate binding proteins, could allow the fine-tuning that provides the temporal ordering of flagellar gene transcription. In this context, we have found that the flgF operon and the distal flgI gene encoding the P-ring, share a sigma 54 activator sequence (class IIA) that differs from the flgH L-ring gene sigma 54 activator site (class IIB) and the hook cluster (class IIC) sigma 54 activator site. The sequential activation of these three subgroups of structural genes reflects the order of assembly of their gene products into the flagellum.

Identification of cis and trans-elements involved in the timed control of a Caulobacter flagellar gene

The genes encoding the structural components of the Caulobacter crescentus flagellum are temporally controlled and their order of expression reflects the sequence of assembly. Transcription of the operon containing the structural gene for the flagellar hook protein occurs at a defined time in the cell cycle, and information necessary for transcription is contained within a region between -81 and -120 base-pairs from the transcription start site. To identify the sequence elements that contribute to the temporal control of hook operon transcription, we constructed deletions and base changes in the 5 region and fused the mutagenized regulatory region to transcription reporter genes. We demonstrate that sequences 3 to the transcription start site do not contribute to temporal control. We confirm that upstream sequences between -81 and -120 base-pairs are necessary for temporal activation, and that transcription also requires sequences at -26 to -46 base-pairs. A specific binding activity for the region between -81 and -122 base-pairs was shown to be temporally controlled, appearing prior to the activation of hook operon transcription. This binding activity was missing from strains containing mutations in flaO and flaW, two genes near the top of the flagellar hierarchy known to be required for hook operon transcription. Thus, the hook operon upstream region contains a sequence element that responds to a temporally controlled trans-acting factor(s), and in concert with a second sequence element causes the timed activation of transcription.

The control of asymmetric gene expression during Caulobacter cell differentiation

The dimorphic bacterium Caulobacter crescentus provides a simple model for cellular differentiation. Each cell division produces two distinct cell types: a swarmer cell and a stalked cell. These cells possess distinet functional morphologies and differential programs of transeription and DNA replication. The synthesis of a single polar flagellum is restricted to the swarmer pole of the predivisional cell by a genetic hierarchy comprising at least 50 genes whose transcription is regulated by novel and ubiquitous promoters, cognate sigma factors, and auxiliary transcriptional regulators. Chromosome replication is restricted to the stalked cell by a unique chromosome origin of replication that may be regulated by a novel cell-specific transcriptional control system. Phosphorylation signals, DNA methylation, differential chromosome structures, protein targeting, and selective protein degradation are also involved in establishing and maintaining cellular asymmetry. The molecular details of these universal cellular processes in C. crescentus will provide paradigms applicable to many general aspects of cellular differentiation.

Expression of cell polarity during Caulobacter differentiation

The bacterium Caulobacter crescentus generates two distinct progeny cells, a motile swarmer cell and a sessile stalked cell, at every cell division. The dramatic morphological and physiological differences between the progeny are expressed in the predivisional cell prior to separation. We review recent work examining mechanisms responsible for differentiation of the incipient swarmer and stalked cell compartments. These include differential transcription of the newly replicated chromosomes, and targeting of proteins to specific poles. The biosynthesis of the polar flagellum is emphasized as a model for studying these processes. Hypotheses concerning the role of the cell poles in expression of asymmetry are discussed.

Dynamic spatial organization of multi-protein complexes controlling microbial polar organization, chromosome replication, and cytokinesis

This project was a program to develop high-throughput methods to identify and characterize spatially localized multiprotein complexes in bacterial cells. We applied a multidisciplinary systems engineering approach to the detailed characterization of localized multi-protein structures in vivo a problem that has previously been approached on a fragmented, piecemeal basis.