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 .

The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the Cl subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.Chromosomal DNA methylation is widespread in prokaryotes and eukaryotes and can affect critical processes such as DNA replication (1, 2), transcription (3-6), and repair of mutational lesions (7). We are examining the regulation and function of a DNA methyltransferase, CcrM*, found in Caulobacter crescentus, a bacterium that undergoes cellular differentiation during each cell cycle (9). Caulobacter chromosomal DNA exhibits cell cycle-dependent patterns of methylation. Constitutive expression of the ccrM gene, yielding chromosomes that are fully methylated throughout the cell cycle, results in an altered developmental program, indicating that variations in methylation state are of regulatory significance (10). Understanding the role of DNA methylation in growth and development has been an elusive goal in many systems. There is abundant evidence correlating the level of cytosine methylation of eukaryotic DNA with gene expression and/or differentiation states (3)(4)(5)(11)(12)(13), but only recently has the role of DNA methylation in eukaryotic organisms been addressed genetically. A deficiency in cytosine methylation results in embryonic lethality in mice (14); in contrast, mutations resulting in deficiencies in DNA methylation in Arabidopsis thaliana (15, 16) and Neurospora crassa (17) are not lethal but cause abnormalities in chromosome segregation behavior. In prokaryotes, the only "regulatory" DNA methyltransferase that has been extensively examined has been the Dam methyltransferase of Escherichia coli and related enterics. Dam methylation is important for temporal control of chromosomal replication (1, 2) and for directing mismatch repair (7) The ccrM (cell-cycle regulated methyltransferase) locus encodes a DNA methyltransferase (CcrM) responsible for N6 methylation of adenine in GANTC sequences (10). In Caulobacter, the single chromosome replicates just once during the cell cycle (19,20). We have shown previously that the remethylation of newly replicated (and thereby hemimethylated) GANTC sites is restricted to the predivisional cell (10), near completion of chromosome replication, and that transcription of the ccrM gene occurs during a similar time fram...

CcrM, an adenine DNA methyltransferase, is essential for viability in Caulobacter crescentus. The CcrM protein is present only in the predivisional stage of the cell cycle, resulting in cell-cycle-dependent variation of the DNA methylation state of the chromosome. The availability of CcrM is controlled in two ways: (1) the ccrM gene is transcribed only in the predivisional cell, and (2) the CcrM protein is rapidly degraded prior to cell division. We demonstrate here that CcrM is an important target of the Lon protease pathway in C. crescentus. In a lon null mutant, ccrM transcription is still temporally regulated, but the CcrM protein is present throughout the cell cycle because of a dramatic increase in its stability that results in a fully methylated chromosome throughout the cell cycle. Because the Lon protease is present throughout the cell cycle, it is likely that the level of CcrM in the cell is controlled by a dynamic balance between temporally varied transcription and constitutive degradation. We have shown previously that restriction of CcrM to the C. crescentus predivisional cell is essential for normal morphogenesis and progression through the cell cycle. Comparison of the lon null mutant strain with a strain whose DNA remains fully methylated as a result of constitutive expression of ccrM suggests that the effect of Lon on DNA methylation contributes to several developmental defects observed in the lon mutant. These defects include a frequent failure to complete cell division and loss of precise cell-cycle control of initiation of DNA replication. Other developmental abnormalities exhibited by the lon null mutant, such as the formation of abnormally long stalks, appear to be unrelated to altered chromosome methylation state. The Lon protease thus exhibits pleiotropic effects in C. crescentus growth and development.

Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation.Caulobacter species are ubiquitous inhabitants of freshwater, marine, and subsurface environments and are sufficiently adaptable to low nutrient conditions that tapwater and even distilled water are ready sources for their isolation (19,48,49). The physiological properties that enable Caulobacter and other oligotrophic species to survive and reproduce in such environments are not well understood. In this work, we used wholegenome transcriptional profiling and genetic analysis to characterize metabolic pathways and transport systems that are differentially expressed by Caulobacter crescentus during growth in three standard laboratory media: a defined "minimal" inorganic salts medium supplemented with either glucose or xylose as the sole carbon source and a complex peptone-yeast extract medium (PYE), in which amino acids serve as the primary carbon source. These observations provide important baseline data on the genetic coordination of metabolism in this model organism.The dimorphic life cycle of C. crescentus has served as a model for understanding prokaryotic development, cell cycle regulation, and asymmetric cell division (24). At each division, C. crescentus divides into two morphologically and physiologically distinct cells: motile swarmer progeny with a single polar flagellum, and sessile stalked progeny displaying an adhesive stalk. The swarmer progeny, in which DNA replication is initially inhibited, is specialized for dispersal ...

The Caulobacter crescentus DNA methyltransferase CcrM (M.CcrMI) methylates the adenine residue in the sequence GANTC. The CcrM DNA methyltransferase is essential for viability, but it does not appear to be part of a DNA restriction-modification system. CcrM homologs are widespread in the alpha subdivision of gramnegative bacteria. We have amplified and sequenced a 258-bp region of the ccrM gene from several of these bacteria, including Rhizobium meliloti, Brucella abortus, Agrobacterium tumefaciens, and Rhodobacter capsulatus. Alignment of the deduced amino acid sequences revealed that these proteins constitute a highly conserved DNA methyltransferase family. Isolation of the full-length ccrM genes from the aquatic bacterium C. crescentus, the soil bacterium R. meliloti, and the intracellular pathogen B. abortus showed that this sequence conservation extends over the entire protein. In at least two alpha subdivision bacteria, R. meliloti and C. crescentus, CcrMmediated methylation has important cellular functions. In both organisms, CcrM is essential for viability. Overexpression of CcrM in either bacterium results in defects in cell division and cell morphology and in the initiation of DNA replication. Finally, the C. crescentus and R. meliloti ccrM genes are functionally interchangeable, as the complemented strains are viable and the chromosomes are methylated. Thus, in both R. meliloti and C. crescentus, CcrM methylation is an integral component of the cell cycle. We speculate that CcrM-mediated DNA methylation is likely to have similar roles among alpha subdivision bacteria.DNA methylation has been identified in both prokaryotes and eukaryotes and has been implicated in many critical cellular processes, including transcriptional regulation (4, 40), initiation of DNA replication (3, 6, 28) and genomic imprinting (33, 34). In mammals and plants, DNA methylation is essential for development. Mice with a null mutation in a cytosine DNA methyltransferase die as embryos (19), and reduced cytosine methylation in Arabidopsis thaliana results in major defects in vegetative and reproductive development (12,35). In prokaryotes, DNA methylation is largely associated with DNA restriction-modification (R/M) systems, where its main function is to allow the cell to differentiate between self and foreign DNA (2, 41). However, bacterial DNA methylation does have other significant biological roles. In Escherichia coli, the Dam DNA methyltransferase (see reference 25 for a recent review), which is not part of an R/M system, regulates several cellular processes, including mismatch repair (15, 24), control of initiation of DNA replication (3,20), and the regulation of gene expression (30,40). Although Dam methylation is involved in a variety of important physiological functions, it is not essential for viability (23). The E. coli Dam methyltransferase is present in several related enteric bacteria, where its function is probably conserved (1).In the bacterium Caulobacter crescentus, the CcrM (M.CcrMI) DNA methyltransferase methyl...

Genetic data suggest that the oligotrophic freshwater bacterium Caulobacter crescentus metabolizes D-xylose through a pathway yielding ␣-ketoglutarate, comparable to the recently described L-arabinose degradation pathway of Azospirillum brasilense. Enzymes of the C. crescentus pathway, including an NAD ؉ -dependent xylose dehydrogenase, are encoded in the xylose-inducible xylXABCD operon (CC0823-CC0819).

Transcription of flagellar genes in Caulobacter crecentus 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.

Principles of modular design are evident in signaling networks that detect and integrate a given signal and, depending on the organism in which the network module is present, transduce this signal to affect different metabolic or developmental pathways. Here we report a global transcriptional analysis of an oxygen sensory͞ signaling network in Caulobacter crescentus consisting of the sensor histidine kinase FixL, its cognate response regulator FixJ, the transcriptional regulator FixK, and the kinase inhibitor FixT. It is known that in rhizobial bacteria these proteins form a network that regulates transcription of genes required for symbiotic nitrogen fixation, anaerobic and microaerobic respiration, and hydrogen metabolism under hypoxic conditions. We have identified a positive feedback loop in this network and present evidence that the negative feedback regulator, FixT, acts to inhibit FixL by mimicking a response regulator. Overall, the core circuit topology of the Fix network is conserved between the rhizobia and C. crescentus, a free-living aerobe that cannot fix nitrogen, respire anaerobically, or metabolize hydrogen. In C. crescentus, the Fix network is required for normal cellular growth during hypoxia and controls expression of genes encoding four distinct aerobic respiratory terminal oxidases and multiple carbon and nitrogen metabolic enzymes. Thus, the Fix network is a conserved sensory͞ signaling module whose transcriptional output has been adapted to the unique physiologies of C. crescentus and the nitrogen-fixing rhizobia.Caulobacter ͉ genetic circuit ͉ hypoxia ͉ signal transduction ͉ two component