Austin Newton

Complete genome sequence of Caulobacter crescentus

The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living α-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

An essential single domain response regulator required for normal cell division and differentiation in Caulobacter crescentus.

Signal transduction pathways mediated by sensor histidine kinases and cognate response regulators control a variety of physiological processes in response to environmental conditions. Here we show that in Caulobacter crescentus these systems also play essential roles in the regulation of polar morphogenesis and cell division. Previous studies have implicated histidine kinase genes pleC and divJ in the regulation of these developmental events. We now report that divK encodes an essential, cell cycle‐regulated homolog of the CheY/Spo0F subfamily and present evidence that this protein is a cognate response regulator of the histidine kinase PleC. The purified kinase domain of PleC, like that of DivJ, can serve as an efficient phosphodonor to DivK and as a phospho‐DivK phosphatase. Based on these and earlier genetic results we propose that PleC and DivK are members of a signal transduction pathway that couples motility and stalk formation to completion of a late cell division cycle event. Gene disruption experiments and the filamentous phenotype of the conditional divK341 mutant reveal that DivK also functions in an essential signal transduction pathway required for cell division, apparently in response to another histidine kinase. We suggest that phosphotransfer mediated by these two‐component signal transduction systems may represent a general mechanism regulating cell differentiation and cell division in response to successive cell cycle checkpoints.

Chromosome replication during development in Caulobacter crescentus.

Abstract Patterns of DNA replication and development have been determined in the dimorphic bacterium Caulobacter crescentus using a new method of cell synchrony. Characteristic DNA cycles were identified for the two cell types. The swarmer cell cycle is composed of G1, S and G2 periods of 65, 85 and 30 minutes, respectively, and the stalked cell cycle is composed of S and G2 periods of 90 and 30 minutes. Thus, the two cell types produced at division initiate DNA synthesis at different times: in the stalked cell chromosome replication begins immediately, while in the swarmer cell the onset of replication is delayed for approximately 65 minutes. Since the pre-synthetic gap in DNA replication corresponds to the time required for stalk formation by the swarmer cell, DNA synthesis is characteristic only of the stalked form of C. crescentus. The results suggest that there may be a structural requirement for initiation of DNA replication in these bacteria, and that in the stalked cell this requirement for initiation has been satisfied at division, while in the swarmer cell further development is required.

Regulation of the Caulobacter flagellar gene hierarchy; not just for motility.

The Caulobacter crescentus flagellum serves not only as a motility apparatus, but also as a key landmark in the differentiation of this asymmetrically dividing bacterium. A distinctive aspect of flagellum biosynthesis is the periodic expression of the flagellar genes during the cell cycle in a sequence corresponding to the order of gene product assembly into the growing flagellum. This program of gene expression is achieved in part by the organization of flagellar genes into a four‐tiered regulatory hierarchy that controls their expression at both the transcriptional and post‐transcriptional levels. Because of the close interconnection of the developmental program to the asymmetric cell‐division cycle in C. crescentus, studies of flagellar gene regulation and motility have also begun to reveal basic mechanisms responsible for control of the cell cycle itself. Here, we review recent work on regulation of the flagellar gene hierarchy in C. crescentus and consider regulatory mechanisms that are distinct from those described in Escherichia coli and Salmonella typhimurium.

Requirement of topoisomerase IV parC and parE genes for cell cycle progression and developmental regulation in Caulobacter crescentus.

We have identified the parC and parE genes encoding DNA topoisomerase IV (Topo IV) in Caulobacter crescentus. We have also characterized the effect of conditional Topo IV mutations on cell division and morphology. Topo IV mutants of C. crescentus are unlike mutants of Escherichia coli and S. typhimurium, which form long filamentous cells that are defective in nucleoid segregation and divide frequently to produce anucleate cells. Topo IV mutants of C. crescentus are highly pinched at multiple sites (cell separation phenotype) and they do not divide to produce cells lacking DNA. These results suggest unique regulatory mechanisms coupling nucleoid partitioning and cell division in this aquatic bacterium. In addition, distinctive nucleoid‐partitioning defects are not apparent in C. crescentus Topo IV mutants as they are in E. coli and S. typhimurium. However, abnormal nucleoid segregation in parE mutant cells could be demonstrated in a genetic background containing a conditional mutation in the C. crescentus ftsA gene, an early cell division gene that is epistatic to parE for cell division and growth. We discuss these results in connection with the possible roles of C. crescentus Topo IV in the regulation of cell division, chromosome partitioning, and late events in polar morphogenesis. Although the ParC and ParE subunits of Topo IV are very similar in sequence to the GyrA and GyrB subunits of DNA gyrase, we have used DNA sequence analysis to identify a highly conserved ‘GyrA box’ sequence that is unique to the GyrA proteins and may serve as a hallmark of the GyrA protein family.

Regulation of flagellin synthesis in the cell cycle of Caulobacter: Dependence on DNA replication

Synthesis of the two filament proteins (flagellin A and flagellin B) of the Caulobacter creascentus flagellum was measured during the cell cycle. Synchronous cells were pulse-labeled with 36S-methionine, and flagellin proteins were isolated from crude extracts by radioimmune precipitation. The results showed that both proteins are maximally induced during the G2 period and that their induction requires de novo transcription. Flagellin A, however, continues to be made in the progeny swarmer cells after flagellin B synthesis has stopped. This discoordination in flagellin A and B synthesis and the relative abundance of the two proteins may result in part from the longer functional half-life of the flagellin A messenger RNA. Analysis of temperature-sensitive DNA chain elongation mutants suggests that the periodicity of flagellin A and B synthesis in the cell cycle is controlled by a late cell cycle event, presumably the completion of chromosome replication.

The Core Dimerization Domains of Histidine Kinases Contain Recognition Specificity for the Cognate Response Regulator

Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.

A set of positively regulated flagellar gene promoters in Caulobacter crescentus with sequence homology to the nif gene promoters of Klebsiella pneumoniae

The study reported here describes nuclease S1 mapping of the in-vivo transcription start sites of transcription units I and III of the hook gene cluster of Caulobacter crescentus. We show that transcription units I and II of this flagellar (fla) gene cluster, which have divergent promoters with transcription start sites separated by 218 nucleotides, are under positive transcriptional control by genes in transcription unit III. The promoters of transcription units I, II, and III were compared with flagellin gene promoters P25, P27 and P29 recently identified in C. crescentus. Promoters PII, P25, and P27, which are under positive regulation by transcription units III to V have strongly conserved sequence elements at -13 and -24 with the consensus sequence (C/T)TGGC(C/G)C-N5-TTGC. The -13, -24 sequence elements are not well conserved in promoter PI, but the promoter does contain a copy of the -13 and -24 consensus sequence 23 base-pairs upstream (PI). The C. crescentus fla gene promoters are not homologous to the canonical Escherichia coli -10, -35 promoter sequence, but they are very similar to the -12, -24 nif gene promoter sequence reported for Klebsiella pneumoniae and Rhizobium sp. The four positively regulated fla gene promoters examined here also share a third conserved element designated II-1, with the consensus sequence C-C-CGGC--AAA--GC-G, located at approximately -100. We speculate that the conserved sequence elements mapping at -13, -24 and -100 are cis-acting regulatory elements required for the transcription and periodic regulation of these fla genes in the C. crescentus cell cycle.

Regulation of periodic protein synthesis in the cell cycle: Control of initiation and termination of flagellar gene expression

Abstract Conditional mutations and drugs that specifically block DNA synthesis and cell division in Caulobacter crescentus have been used to analyze the role of the cell cycle in differentiation. The results suggest that the DNA synthetic pathway, and not an intact cell division pathway, acts to time the periodic synthesis of flagellin A, flagellin B and the hook protein. The expression of these flagellar genes is apparently controlled at the level of transcription, with initiation of the synthetic periods dependent on a specific stage of chromosome replication. The required stage of replication, or DNA execution point, occurs near mid-S phase at approximately 0.65 of the swarmer cell cycle. Examination of a C. crescentus polyhook mutant PCM103 indicates, however, that the DNA synthetic pathway is only one level of regulation over flagellum formation. In the mutant, hook protein synthesis is coupled to DNA synthesis and is initiated at the expected time in the cell cycle, but hook protein synthesis is not repressed at the normal time, hook assembly fails to terminate and only a low level of flagellin is detected. Thus the defect in this strain, which grows and divides normally, appears to affect a sequence of developmental events at a point after the induction of the hook protein. These findings are consistent with a hierarchy of controls that regulates the periodic synthesis of flagellar proteins and their assembly in the flagellum. Initiation of hook protein and flagellin synthesis requires a specific stage of chromosome replication, while later stages of flagellum formation, including the termination of hook protein synthesis, also require a second level of regulation that is defective in strain PCM 103.

Temporal control of the cell cycle in Caulobacter crescentus: Roles of DNA chain elongation and completion

The functional relationships between cell cycle steps in Caulobacter crescentus have been examined by reciprocal shift experiments ( Hereford & Hartwell, 1974 ; Jarvick & Botstein, 1973 ) using temperature-sensitive mutations ( Osley & Newton, 1977 ) and drugs that reversibly block specific cell cycle steps. Hydroxyurea was used to inhibit DNA chain elongation (DNA e ) and penicillin was used to block the initiation of cell division (DIV i ; Terrana & Newton 1976 ). These experiments show that the cell cycle of Caulobacter can be organized into at least two dependent pathways. One of these pathways mediates the sequence of DNA events, DNA i (initiation of chain elongation)→DNA e →DNA c (completion of chain elongation), and the other pathway mediates the sequence of division events, DIV i →DIV p (progressive pinching at the division site)→CS (cell separation). Shift experiments in synchronous cultures showed that the initiation and completion of the cell division pathways require two successive stages of the DNA synthetic pathway: DIV i steps are dependent on DNA e and CS steps are dependent on DNA c . These and earlier results suggest that chromosome replication may act as a cell cycle “clock” to time events in the cell division pathway and to control the periodic expression of genes required for formation of the flagellum ( Osley et al. , 1977 ) and other developmental structures.