In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H2O2 and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.

Summary A transposon Tn5 mutagenesis library was generated from Caulobacter crescentus strain NA1000, and clones with deficiency in survival in a high concentration of NaCl were selected. One of these clones, 37G10, has the Tn5 integrated within the coding region of the transcription termination factor Rho. Analysis of this mutant phenotype showed that the cells are motile and present a normal cell cycle, but have a longer generation time. This strain is sensitive to acidic pH, to the presence of different salts and to heat shock, but it responds well to UV light and alkaline pH. The most striking phenotype of the rho mutant is that it is extremely sensitive to oxidative stress, in both exponential and stationary phases. Experiments using a transcriptional fusion of the rho promoter region to the lacZ gene showed that rho gene expression varies during the cell cycle, showing very low expression levels at the swarmer cell stage and presenting maximum levels in early predivisional cells. Transcription of the rho gene is increased in the rho mutant strain, which is indicative of an autoregulatory circuit, and there is a small variation in the cell cycle pattern of expression. Several peptides have their synthesis altered in the mutant strain, as analysed by two‐dimensional gel electrophoresis, most of which show a reduction in expression. These results indicate that the Rho factor is essential for an efficient response to certain stresses in Caulobacter.

Most organisms that grow in the presence of oxygen possess catalases and/or peroxidases, which are necessary for scavenging the H 2 O 2 produced by aerobic metabolism. In this work we investigate the pathways that regulate the Caulobacter crescentus katG gene, encoding the only enzyme with catalase-peroxidase function in this bacterium. The transcriptional start site of the katG gene was determined, showing a short 5 untranslated region. The katG regulatory region was mapped by serial deletions, and the results indicate that there is a single promoter, which is responsible for induction at stationary phase. An oxyR mutant strain was constructed; it showed decreased katG expression, and no KatG protein or catalase-peroxidase activity was detected in stationary-phase cell extracts, implying that OxyR is the main positive regulator of the C. crescentus katG gene. Purified OxyR protein bound to the katG regulatory region between nucleotides ؊42 and ؊91 from the transcription start site, as determined by a DNase I footprinting assay, and a canonical OxyR binding site was found in this region. Moreover, OxyR binding was shown to be redox dependent, given that only oxidized proteins bound adjacent to the ؊35 sequence of the promoter and the katG P1 promoter was activated by OxyR in an H 2 O 2 -dependent manner. On the other hand, this work showed that the iron-responsive regulator Fur does not regulate C. crescentus katG, since a fur mutant strain presented wild-type levels of katG transcription and catalase-peroxidase production and activity, and the purified Fur protein was not able to bind to the katG regulatory region.

An attempt to characterize Caulobacter crescentus genes important for the response to high concentrations of NaCl was initiated by the isolation of mutants defective in survival in the presence of 85 mM NaCl. A transposon Tn5 library was screened, and five strains which contained different genes disrupted by the transposon were isolated. Three of the mutants had the Tn5 in genes involved in lipopolysaccharide biosynthesis, one had the Tn5 in the nhaA gene, which encodes a Na ؉ /H ؉ antiporter, and one had the Tn5 in the ppiD gene, which encodes a peptidyl-prolyl cis-trans isomerase. All the mutant strains showed severe growth arrest in the presence of 85 mM NaCl, but only the nhaA mutant showed decreased viability under these conditions. All the mutants except the nhaA mutant showed a slightly reduced viability in the presence of 40 mM KCl, but all the strains showed a more severe reduction in viability in the presence of 150 mM sucrose, suggesting that they are defective in responding to osmotic shock. The promoter regions of each disrupted gene were cloned in lacZ reporter vectors, and the pattern of expression in response to NaCl and sucrose was determined; this showed that both agents induced ppiD and nhaA gene expression but did not induce the other genes. Furthermore, the ppiD gene was not induced by heat shock, indicating that it does not belong to the 32 regulon, as opposed to what was observed for its Escherichia coli homolog.

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The impossibility of obtaining a rho null mutant and sensitivity to bicyclomycin have indicated that rho is essential for the viability of Caulobacter crescentus. Transcription gene fusions of sequences with serial deletions of the rho 5 untranslated region (5-UTR) with a lacZ reporter gene indicated that rho is autoregulated at the level of attenuation of transcription in the 5-UTR.The importance of Rho-dependent termination in bacterial gene expression is well established for genes that are regulated by termination of transcription in the leader sequences, as described for the tna operon from Escherichia coli (15,33,35). Studies with E. coli, Rhodobacter sphaeroides, and Micrococcus luteus showed that rho is an essential gene in these bacteria (6,8,26) but not in Bacillus subtilis and Staphylococcus aureus (29, 34). The current model proposes that Rho loads onto nascent mRNAs at a cytosine-rich region that lacks a strong secondary structure, known as the Rho utilization (rut) site (1,23,30). It has been proposed that once bound, a hexameric Rho complex hydrolyzes ATP, moves directionally 5Ј-3Ј along the RNA to the site of transcription, and with its helicase function, disengages the message from the paused RNA polymerase and the DNA template, separating the transcription complex (4,17,22,27,31,32). The Rho factor was described to be autogenously regulated in E. coli and B. subtilis via an attenuation mechanism that occurs in Rho-dependent terminators located within the mRNA leader of the rho gene (2,12,20). The ATPase activity of Rho in several bacteria has been described and characterized (10,11,12,16,25), but autoregulation by Rho-dependent terminators in bacteria other than E. coli and B. subtilis is yet to be characterized.In a previous work from our group, a rho mutation generated by a Tn5 insertion (strain SP3710) caused deficiency in survival to high salt concentration and acidic pH and resulted in an extreme sensitivity to oxidative stress (13). Experiments showed that transcription of the C. crescentus rho gene increases in the rho mutant strain, which is indicative of a negative autoregulatory circuit (13).The Rho factor is essential for C. crescentus. The Rho protein in SP3710 was interrupted after residue Gly-112, immediately before the first RNA binding motif (13). Immunoblotting assays of cell extracts probed with a polyclonal Rho antiserum showed that SP3710 still produces a 45-kDa truncated polypeptide but does not present the complete form of the Rho protein of the parental strain, NA1000, except when it is complemented in trans with the rho gene (Fig. 1A). The C. crescentus Rho protein has a predicted molecular mass of 52.8 kDa but migrates with an apparent mass of 60 kDa in sodium dodecyl sulfate-polyacrylamide gel. The 45-kDa protein should

Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. ␤-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

The entry into stationary phase causes a change in the pattern of gene expression of bacteria, when the cells must express a whole set of genes involved mainly with resistance to starvation and to environmental stresses. As an attempt to identify genes important for the survival of Caulobacter crescentus in stationary phase, we have screened a library of 5,000 clones generated by random transposon mutagenesis for mutants that showed reduced viability after prolonged growth. Four clones were selected, which displayed either lower viability or a longer time of recovery from stationary phase. The genes disrupted were identified, and the gene products were found to be mainly involved with amino acid metabolism (glutamate N-acetyltransferase, 4-hydroxyphenylpyruvate dioxygenase and L-aspartate oxidase) or with recombination (exonuclease RecJ). Each mutant was tested for resistance to stresses, such as oxidative, saline, acidic, heat and UV exposure, showing different responses. Although the mutations obtained were not in genes involved specifically in stationary phase, our results suggest that amino acids metabolism may play an important role in keeping viability during this growth phase.