Past studies of bacterial metabolism have primarily focused on cells adapted to high nutrient conditions. In this work, in contrast, we analyzed the metabolism of an oligotrophic organism, Caulobacter crescentus
. Oligotrophs thrive where nutrients are perpetually scarce, the usual situation in most aquatic and terrestrial habitats on earth. An improved understanding of oligotrophs' metabolic activities is essential to understanding biogeochemical processes that have major impact on the environment (20
). We describe here a comparison of gene expression during growth of C. crescentus
on common laboratory media. These data provide insights into C. crescentus
metabolic pathways and regulatory strategies and establish a baseline for future experiments with these media. We assessed expression patterns for over 80% of the known genes in the C. crescentus
genome in each pairwise medium comparison and found that over 12% of the genes examined showed significant differences in expression in at least one medium comparison. Not surprisingly, most of the differences occurred between the complex PYE medium and the M2 minimal salts media regardless of the carbon source used in the M2 medium. Fewer differences were seen between the two M2 media, but the differences that were observed may identify transporters, enzymes, and regulators specifically relevant to glucose and xylose utilization.
The Entner-Doudoroff pathway has been shown to be active in C. crescentus
during growth on glucose (48
). In the data presented here, all of the genes encoding enzymes of the Entner-Doudoroff pathway were found to be significantly induced during growth on glucose-containing media compared to PYE. Mutations in three genes (CC1495, CC2054, and CC2055) encoding enzymes of the Entner-Doudoroff pathway rendered C. crescentus
incapable of growth with glucose as the sole carbon source. The Entner-Doudoroff pathway is thus essential for glucose utilization in C. crescentus
, a situation previously seen only in Pseudomonas aeruginosa
) and Burkholderia cepacia
). In contrast, in several free-living α-proteobacteria in which glucose metabolism has been examined, both the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways are used (3
The apparent absence of the enzyme phosphofructokinase in C. crescentus
would preclude use of the upper branch of the Embden-Meyerhof-Parnas pathway. Thus, instead of generating glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, glucose is processed into glyceraldehyde-3-phosphate and pyruvate. There appears to be little transcriptional regulation of the genes encoding enzymes for the latter steps of glycolysis (taking glyceraldehyde-3-phosphate to pyruvate), as mRNA levels showed minimal differences between media. In contrast, in E. coli
, expression of all genes in the lower branch of the Embden-Meyerhof-Parnas pathway except pykA
(pyruvate kinases) increases during fermentation on glucose compared to fermentation on xylose (16
). The E. coli
gene expression data agree with metabolic flux estimates in that all steps in the lower part of the glycolytic pathway are expected to have higher flux during fermentation of glucose compared to fermentation of xylose except for the conversion of phosphoenolpyruvate to pyruvate (16
). Whether similar expression of glycolytic genes during growth of C. crescentus
on xylose and glucose likewise reflects comparable carbon flux through the lower part of the glycolytic pathway during growth on glucose and xylose is uncertain, given that the route of xylose catabolism is unknown.
There are many genes whose products have the same predicted enzymatic function in the current C. crescentus
genome annotation but which exhibited distinct expression patterns in the microarray data (Table S2). Some differentially regulated isozymes may participate in distinct pathways that are differentially induced depending on the medium. For example, in an oligotrophic Spirillum
sp., the relative activities of three forms of lactate dehydrogenase vary as a function of the lactate concentration in the growth medium (37
). The exact roles of many of the C. crescentus
isozymes remain to be determined. In such cases, differential regulation may provide clues to distinct functions.
One example is particularly relevant to glucose catabolism. Two genes (CC0784 and CC1495) are annotated as encoding KDPG aldolase, which catalyzes the final step in the Entner-Doudoroff pathway. Expression of CC1495 is elevated in M2 minimal media with either xylose or glucose as the carbon source compared to PYE, whereas expression of CC0784 is unchanged. Subsequent mutational results demonstrated that CC1495 is essential for growth on glucose, implying that CC0784 (assuming that it is expressed) is not capable of functionally substituting for CC1495 in glucose catabolism. On the other hand, the CC1495 mutant strains could still form colonies with galactose as the carbon source. The CC0784 polypeptide sequence resembles gene products annotated as 2-dehydro-3-deoxyphosphogalactonate aldolases from several organisms, including Ralstonia solanacearum
, Bradyrhizobium japonicum
, and Brucella
species; this resemblance is considerably stronger than that shown by the CC1495 polypeptide. It has been reported previously that C. crescentus
may use some version of the Entner-Doudoroff pathway for galactose catabolism (29
). Our results suggest that the CC0784 product, rather than participating in the glucose Entner-Doudoroff pathway, may be involved in a version of the Entner-Doudoroff pathway dedicated to galactose.
The pathway of xylose degradation in C. crescentus
may be distinct from known pathways in other microbes, as genes encoding key enzymes for those pathways (particularly xylose isomerase and xylulose kinase) are not apparent in the C. crescentus
genome. An inducible xylose dehydrogenase activity has been demonstrated in C. crescentus
, which may serve as the initial step in xylose catabolism (48
). The only known C. crescentus
mutant strain that is unable to grow on xylose resulted from a Tn5
insertion in the xylX
(CC0823) gene (39
). This insertion showed strongly xylose-inducible β-galactosidase activity. Microarray data presented here confirm xylose induction of CC0823, which turns out to be the first gene in a potential five-gene operon (CC0823 to CC0819), the largest transcription unit whose expression responds to xylose.
The CC0823 product does not resemble any gene of known function, but it is homologous to genes in other genomes, including Mesorhizobium loti
, Streptomyces coelicolor
, Agrobacterium tumefaciens
, Pseudomonas putida
, Bradyrhizobium japonicum
, and Sinorhizobium meliloti
. The xyl
mutant phenotype of the Tn5
insertion could reflect a critical role for the CC0823 product in xylose metabolism (39
); alternatively, the xyl
mutant phenotype could result from polar effects of the insertion on expression of critical downstream genes in the operon. Two putative dehydrogenases (CC0822 and CC0821) of unknown substrate specificity are encoded in this operon, either of which could potentially be responsible for inducible xylose dehydrogenase activity.
The ability to replenish intermediates of the tricarboxylic acid cycle can be critical for growth of microbes on a single carbon source (17
). Under such conditions, tricarboxylic acid cycle intermediates are continually siphoned off for various biosynthetic processes, compromising continued flux through the pathway. Expression data and genetic analysis suggest that carbon flux through the tricarboxylic acid cycle is managed differently during growth on glucose and xylose. The expression of phosphoenolpyruvate carboxylase is induced during growth on glucose but not xylose, and genetic evidence is presented above that supplementation of the tricarboxylic acid cycle with oxalacetate via phosphoenolpyruvate carboxylase is critical for growth on glucose but not xylose.
In contrast, expression of isocitrate lyase (CC1764) is specifically elevated during growth on xylose. Isocitrate lyase catalyzes the conversion of isocitrate to glyoxylate and succinate, initiating the glyoxylate bypass. The succinate generated by this process can be drawn off for other biosynthetic pathways. Glyoxylate, meanwhile, can be combined with acetyl-coenzyme A by malate synthase to produce malate, allowing a modified tricarboxylic acid cycle to continue. Interestingly, expression of malate synthase, encoded by the gene (CC1765) adjacent to that encoding isocitrate lyase, was not observed to increase coordinately in response to xylose in the microarrays. Future studies are warranted to examine the role of the glyoxylate bypass during growth on xylose and other carbon sources.
The ability to simultaneously import a variety of growth substrates could be critical for survival in oligotrophic environments (21
). As such, it seems unlikely that all of the putative transporters and exoenzymes whose expression is induced by xylose are utilized solely, or are even necessary, for the import and utilization of this sugar. The CC0505 gene, for example, is strongly induced by xylose, but a CC0505 knockout strain shows no defects in growth on xylose (data not shown). We hypothesize that C. crescentus
uses xylose as an indicator that metabolites derived from the breakdown of plant cell walls are available. Other microorganisms, particularly soil microbes such as Streptomyces
species, are known to use small molecules resulting from extracellular polymer breakdown as a positive feedback signal to induce degradative enzymes (22
). Similarly, growth of Thermotoga maritima
on xylose induces genes for two xylanases and an α-glucosidase, and growth of T. maritima
on xylan additionally increases expression of genes for an α-glucuronidase and a β-d
Since xylose in natural habitats would presumably be acquired by breakdown of xylan, xylose may be used to trigger the expression of extracellular hydrolytic enzymes relevant to breakdown of lignicellulose (of which xylan is a major constituent) as well as the enzymes needed for the catabolism of xylose itself, as in the fungus Aspergillus niger
). Environments rich in xylan should contain cellulose as well; interestingly, however, xylose does not induce expression of endoglucanases or β-glucosidases that could (at least theoretically) be used by C. crescentus
for cellulose degradation.
The abundance of TonB-dependent receptors induced by xylose could provide cells with the ability to import products of extracellular lignocellulose degradation, but it remains to be determined what metabolites each transporter is capable of bringing into the periplasm. In an effort to further characterize the transporters induced by xylose, we used BLASTP to compare xylose-induced C. crescentus
transporters to the NCBI database. Somewhat surprisingly, many of the strongest homologies were to proteins encoded by Xanthomonas campestris
, a plant pathogen that secretes exoenzymes to attack plant cell walls. The virulence of at least one Xanthomonas
species (X. oryzae
, which causes rice blast disease) is dependent on the ability to secrete xylanase (51
). The X. campestris
genome contains bidirectional best hits to four of the xylose-induced TonB-dependent receptors (CC0442, CC0999, CC2832, and CC3336) as well as the putative inner membrane xylose transporter (CC0814) and the SapC peptide permease homolog (CC1000).
are not close phylogenetically, their genomic contents bear intriguing similarities. In addition to having sets of putative exoenzymes similar to C. crescentus
, the sequenced genomes of the Xanthomonas
species X. campestris
and X. citri
also display an abundance (nearly 50) of TonB-dependent receptors (10
). There are also hints that regulation by xylose may be similar in X. campestris
and C. crescentus
. The X. campestris
genome has several close matches to the DNA sequence motif associated with xylose regulation in C. crescentus
(Table ), with three of the four strongest instances of this motif in the X. campestris
genome located upstream of putative transcription units containing xylose-associated genes (xylose isomerase, xylosidase, and xylanase) (Table S5). Thus, although C. crescentus
has not been reported to act as a plant pathogen, it may have mechanisms for acquiring plant polymer-derived metabolites similar to those found in Xanthomonas
Environmentally responsive signal transduction pathways in C. crescentus have not been extensively explored. The collection of large microarray data sets, as presented here, provides some initial insight into regulatory responses to nutrient levels in this organism. A set of genes whose expression responds to the availability of xylose have been identified, along with a cis-acting site that appears to represent the target for a xylose-responsive repressor. Two potential regulatory sites have also been identified for glucose-responsive genes; these need to be tested, and the trans-acting factors controlling xylose and glucose regulation will be pursued in future work. Coregulation of many genes and potential operons encoding amino acid synthesis or degradation functions has been observed.
Future work will continue to characterize the traits that allow C. crescentus
to be a successful oligotroph by exploring this organism's metabolism in more dilute media, in mixtures of substrates, and in continuous cultures. Finally, the stage is set for exploring the interface between environmental and cell cycle regulation. What environmental cues influence cell cycle progression? Are there cell cycle checkpoints that determine if a cell has sufficient resources to proceed with division? How are individual promoters influenced by both the environmental and cell cycle state? As just one example, expression of the glycine cleavage system (gcv
) genes (CC3355 to CC3352) is strongly induced in PYE medium versus M2, presumably due to relatively high levels of exogenous glycine. During the cell cycle, expression of the gcv
genes peaks in swarmer cells and then is repressed dramatically in predivisional cells (32
). Is the same transcription factor used in both forms of regulation? What controls cell cycle regulation of this promoter, and why? Further application of molecular and classical genetic approaches with microarray analysis can provide insight into such questions.