The Nucleoid-Associated Protein GapR Uses Conserved Structural Elements To Oligomerize and Bind DNA.

Nucleoid-associated proteins (NAPs) are DNA binding proteins critical for the organization and function of the bacterial chromosome. A newly discovered NAP in Caulobacter crescentus, GapR, is thought to facilitate the movement of the replication and transcription machines along the chromosome by stimulating type II topoisomerases to remove positive supercoiling. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding. Moreover, we show that GapR is maintained as a tetramer upon its dissociation from DNA and that tetrameric GapR is capable of binding DNA molecules in vitro Analysis of protein chimeras revealed that two helices of GapR are functionally conserved in H-NS, demonstrating that two evolutionarily distant NAPs with distinct mechanisms of action utilize conserved structural elements to oligomerize and bind DNA.IMPORTANCE Bacteria organize their genetic material in a structure called the nucleoid, which needs to be compact to fit inside the cell and, at the same time, dynamic to allow high rates of replication and transcription. Nucleoid-associated proteins (NAPs) play a pivotal role in this process, so their detailed characterization is crucial for our understanding of DNA organization into bacterial cells. Even though NAPs affect DNA-related processes differently, all of them have to oligomerize and bind DNA for their function. The significance of this study is the identification of structural elements involved in the oligomerization and DNA binding of a newly discovered NAP in C. crescentus and the demonstration that structural elements are conserved in evolutionarily distant and functionally distinct NAPs.

Transcriptomic analysis of the stationary phase response regulator SpdR in Caulobacter crescentus.

BACKGROUND: As bacterial cells enter stationary phase, they adjust their growth rate to comply with nutrient restriction and acquire increased resistance to several stresses. These events are regulated by controlling gene expression at this phase, changing the mode of exponential growth into that of growth arrest, and increasing the expression of proteins involved in stress resistance. The two-component system SpdR/SpdS is required for the activation of transcription of the Caulobacter crescentus cspD gene at the onset of stationary phase. RESULTS: In this work, we showed that both SpdR and SpdS are also induced upon entry into stationary phase, and this induction is partly mediated by ppGpp and it is not auto-regulated. Global transcriptional analysis at early stationary phase of a spdR null mutant strain compared to the wild type strain was carried out by DNA microarray. Twenty-three genes showed at least twofold decreased expression in the spdR deletion mutant strain relative to its parental strain, including cspD, while five genes showed increased expression in the mutant. The expression of a set of nine genes was evaluated by quantitative real time PCR, validating the microarray data, and indicating an important role for SpdR at stationary phase. Several of the differentially expressed genes can be involved in modulating gene expression, including four transcriptional regulators, and the RNA regulatory protein Hfq. The ribosomal proteins NusE and NusG, which also have additional regulatory functions in transcription and translation, were also downregulated in the spdR mutant, as well as the ParE1 toxin. The purified SpdR protein was shown to bind to the regulatory region of CC0517 by Electrophoretic Mobility Shift Assay, and the SpdR-regulated gene CC0731 was shown to be expressed at a lower level in the null cspD mutant, suggesting that at least part of the effect of SpdR on the expression of this gene is indirect. CONCLUSIONS: The results indicate that SpdR regulates several genes encoding proteins of regulatory function, which in turn may be required for the expression of other genes important for the transition to stationary phase.

CspC regulates the expression of the glyoxylate cycle genes at stationary phase in Caulobacter.

BACKGROUND: The Cold Shock proteins are RNA binding proteins involved in various cellular processes, including adaptation to low temperature, nutritional stress, cell growth and stationary phase. They may have an impact on gene expression by interfering with RNA stability and acting as transcription antiterminators. Caulobacter crescentus cspC is an essential gene encoding a stationary phase-induced protein of the Cold Shock Protein family and this work had as goal investigating the basis for the requirement of this gene for survival at this phase. In this work we investigate the role of CspC in C. crescentus stationary phase and discuss the molecular mechanisms that could be involved. RESULTS: The expression of cspC increased significantly at stationary phase in complex media and in glucose depletion, indicating a putative role in responding to carbon starvation. Global transcriptional profiling experiments comparing cspC and the wild type strain both at exponential and stationary phases as well as comparing exponential and stationary phase in wild type strain were carried out by DNA microarray analysis. The results showed that the absence of cspC affected the transcription of 11 genes at exponential phase and 60 genes at stationary phase. Among the differentially expressed genes it is worth noting those encoding respiratory enzymes and genes for sulfur metabolism, which were upregulated, and those encoding enzymes of the glyoxylate cycle, which were severely downregulated in the mutant at stationary phase. mRNA decay experiments showed that the aceA mRNA, encoding isocitrate lyase, was less stable in the cspC mutant, indicating that this effect was at least partially due to posttranscriptional regulation. These observations were supported by the observed arrested growth phenotype of the cspC strain when grown in acetate as the sole carbon source, and by the upregulation of genes for assimilatory sulfate reduction and methionine biosynthesis. CONCLUSIONS: The stationary phase-induced RNA binding protein CspC has an important role in gene expression at this phase, and is necessary for maximal expression of the glyoxylate cycle genes. In the case of aceA, its downregulation may be attributed to the shorter half-life of the mRNA in the cspC mutant, indicating that one of the possible regulatory mechanisms is via altering RNA stabilization.

A comprehensive genomic, transcriptomic and proteomic analysis of a hyperosmotic stress sensitive α-proteobacterium.

BACKGROUND: With the aim of remaining viable, bacteria must deal with changes in environmental conditions, including increases in external osmolarity. While studies concerning bacterial response to this stress condition have focused on soil, marine and enteric species, this report is about Caulobacter crescentus, a species inhabiting freshwater oligotrophic habitats. RESULTS: A genomic analysis reported in this study shows that most of the classical genes known to be involved in intracellular solute accumulation under osmotic adaptation are missing in C. crescentus. Consistent with this observation, growth assays revealed a restricted capability of the bacterium to propagate under hyperosmotic stress, and addition of the compatible solute glycine betaine did not improve bacterial resistance. A combination of transcriptomic and proteomic analyses indicated quite similar changes triggered by the presence of either salt or sucrose, including down-regulation of many housekeeping processes and up-regulation of functions related to environmental adaptation. Furthermore, a GC-MS analysis revealed some metabolites at slightly increased levels in stressed cells, but none of them corresponding to well-established compatible solutes. CONCLUSION: Despite a clear response to hyperosmotic stress, it seems that the restricted capability of C. crescentus to tolerate this unfavorable condition is probably a consequence of the inability to accumulate intracellular solutes. This finding is consistent with the ecology of the bacterium, which inhabits aquatic environments with low nutrient concentration.

Proteomic analysis of yeast mutant RNA exosome complexes.

The yeast exosome is a conserved multiprotein complex essential for RNA processing and degradation. The complex is formed by a nine-subunit core that associates with two hydrolytic 3'-5' exoribonucleases. Although catalytically inert, the assembly of this nine-subunit core seems to be essential for the exosome activity, as mutations in regions that do not directly bind RNA or are not in the active sites of the exonucleases impair the function of the complex. Previously isolated mutations in the exosome core subunit Rrp43p have been shown to negatively affect the function of the complex. With the aim of investigating the effect of these mutations on the complex stability and activity, Rrp43p and its mutant forms were purified by means of the TAP method. Mass spectrometry analyses showed that lower amounts of the exosome subunits are copurified with the mutant Rrp43p proteins. Additionally, by decreasing the stability of the exosome, other nonspecific protein interactions are favored (the data have been deposited to the ProteomeXchange with identifier PXD000580). Exosome copurified with mutant Rrp43p exhibited increased exonuclease activity, suggesting higher dissociation constants for these mutant complexes. Therefore, data reported here indicate that complexes containing a mutant Rrp43p exhibit decreased stability and provide information on additional protein interactions.

Global transcriptional response of Caulobacter crescentus to iron availability.

BACKGROUND: In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater α-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. RESULTS: In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identified 42 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB-dependent receptor gene clusters, and feoAB), riboflavin biosynthesis and genes encoding hypothetical proteins. Most of these genes are associated with predicted Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by ß-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 27 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, included in this group are also TonB-dependent receptors genes and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of 113 more genes, including induction of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as repression of genes implicated in amino acid metabolism, chemotaxis and motility. CONCLUSIONS: Using a global transcriptional approach, we determined the C. crescentus iron stimulon. Many but not all of iron responsive genes were directly or indirectly controlled by Fur. The iron limitation stimulon overlaps with other regulatory systems, such as the RpoH and FixK regulons. Altogether, our results showed that adaptation of C. crescentus to iron limitation not only involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins, but also includes novel genes and regulatory mechanisms.

Extracytoplasmic function (ECF) sigma factor σF is involved in Caulobacter crescentus response to heavy metal stress.

BACKGROUND: The α-proteobacterium Caulobacter crescentus inhabits low-nutrient environments and can tolerate certain levels of heavy metals in these sites. It has been reported that C. crescentus responds to exposure to various heavy metals by altering the expression of a large number of genes. RESULTS: In this work, we show that the ECF sigma factor σF is one of the regulatory proteins involved in the control of the transcriptional response to chromium and cadmium. Microarray experiments indicate that σF controls eight genes during chromium stress, most of which were previously described as induced by heavy metals. Surprisingly, σF itself is not strongly auto-regulated under metal stress conditions. Interestingly, σF-dependent genes are not induced in the presence of agents that generate reactive oxygen species. Promoter analyses revealed that a conserved σF-dependent sequence is located upstream of all genes of the σF regulon. In addition, we show that the second gene in the sigF operon acts as a negative regulator of σF function, and the encoded protein has been named NrsF (Negative regulator of sigma F). Substitution of two conserved cysteine residues (C131 and C181) in NrsF affects its ability to maintain the expression of σF-dependent genes at basal levels. Furthermore, we show that σF is released into the cytoplasm during chromium stress and in cells carrying point mutations in both conserved cysteines of the protein NrsF. CONCLUSION: A possible mechanism for induction of the σF-dependent genes by chromium and cadmium is the inactivation of the putative anti-sigma factor NrsF, leading to the release of σF to bind RNA polymerase core and drive transcription of its regulon.

A two-component system, an anti-sigma factor and two paralogous ECF sigma factors are involved in the control of general stress response in Caulobacter crescentus.

The extracytoplasmic function sigma factor σ(T) is the master regulator of general stress response in Caulobacter crescentus and controls the expression of its paralogue σ(U). In this work we showed that PhyR and NepR act, respectively, as positive and negative regulators of σ(T) expression and function. Biochemical data also demonstrated that NepR directly binds σ(T) and the phosphorylated form of PhyR. We also described the essential role of the histidine kinase gene CC3474, here denominated phyK, for expression of σ(T)-dependent genes and for resistance to stress conditions. Additionally, in vivo evidence of PhyK-dependent phosphorylation of PhyR is presented. This study also identified a conserved cysteine residue (C95) located in the periplasmic portion of PhyK that is crucial for the function of the protein. Furthermore, we showed that PhyK, PhyR and σ(T) regulate the same set of genes and that σ(T) apparently directly controls most of its regulon. In contrast, σ(U) seems to have a very modest contribution to the expression of a subset of σ(T)-dependent genes. In conclusion, this report describes the molecular mechanism involved in the control of general stress response in C. crescentus.

The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the human pathogen Cryptococcus neoformans.

Nitrogen uptake and metabolism are essential to microbial growth. Gat1 belongs to a conserved family of zinc finger containing transcriptional regulators known as GATA-factors. These factors activate the transcription of Nitrogen Catabolite Repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. Cryptococcus neoformans GAT1 is an ortholog to the Aspergillus nidulans AreA and Candida albicans GAT1 genes. In an attempt to define the function of this transcriptional regulator in C. neoformans, we generated null mutants (gat1Δ) of this gene. The gat1 mutant exhibited impaired growth on all amino acids tested as sole nitrogen sources, with the exception of arginine and proline. Furthermore, the gat1 mutant did not display resistance to rapamycin, an immunosuppressant drug that transiently mimics a low-quality nitrogen source. Gat1 is not required for C. neoformans survival during macrophage infection or for virulence in a mouse model of cryptococcosis. Microarray analysis allowed the identification of target genes that are regulated by Gat1 in the presence of proline, a poor and non-repressing nitrogen source. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule biosynthesis, in addition to NCR-sensitive genes, are Gat1-regulated in C. neoformans.

The transcriptional response to cadmium, organic hydroperoxide, singlet oxygen and UV-A mediated by the sigmaE-ChrR system in Caulobacter crescentus.

Caulobacter crescentussigma(E) belongs to the ECF (extracytoplasmic function) subfamily of RNA polymerase sigma factors, whose members regulate gene expression in response to distinct environmental stresses. During physiological growth conditions, data indicate that sigma(E) is maintained in reduced levels due to the action of ChrR, a negative regulator of rpoE gene expression and function. However, once bacterial cells are exposed to cadmium, organic hydroperoxide, singlet oxygen or UV-A irradiation, transcription of rpoE is induced in a sigma(E)-dependent manner. Site-directed mutagenesis indicated that residue C188 in ChrR is critical for the cadmium response while residues H140 and H142 are required for the bacterial response to organic hydroperoxide, singlet oxygen and UV-A. Global transcriptional analysis showed that sigma(E) regulates genes involved in protecting cells against oxidative damages. A combination of transcriptional start site identification and promoter prediction revealed that some of these genes contain a putative sigma(E)-dependent motif in their upstream regions. Furthermore, deletion of rpoE and two sigma(E)-dependent genes (cfaS and hsp20) impairs Caulobacter survival when singlet oxygen is constantly generated in the cells.

The ECF sigma factor sigma(T) is involved in osmotic and oxidative stress responses in Caulobacter crescentus.

Sigma factors of the ECF subfamily are important regulators of stress responses in bacteria. Analysis of Caulobacter crescentus genome sequence has indicated the presence of 13 members of the ECF (extracytoplasmic function) subfamily, suggesting that these regulators play an important role in C. crescentus physiology. This work describes the characterization of two highly similar C. crescentus ECF sigma factors, sigma(U) and sigma(T). The corresponding genes are not essential under normal growth conditions and absence of sigma(U) does not impair bacterial resistance to the environmental stresses tested. However, absence of sigma(T) significantly affects the ability of C. crescentus cells to survive osmotic and oxidative stress. Using transcription fusions to sigT and sigU upstream regions we demonstrate that both genes are induced by osmotic stress in a sigma(T)-dependent manner. Determination of sigU and sigT transcription start sites revealed an identical promoter motif, typical of ECF-dependent promoters. Transcriptome analysis revealed 40 putative members of the sigma(T) regulon, including sigU and sigR, encoding another ECF subfamily member, and genes involved in general stress responses and cell envelope functions. Twenty of those genes exhibit the sigT/sigU promoter motif in their upstream regions. Our data indicate a role of sigma(T) in distinct stress responses in C. crescentus.