Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon

<p>Abstract</p> <p>Background</p> <p><it>Xylella fastidiosa</it>, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, <it>X. fastidiosa </it>needs to cope with nitrogen limitation in its natural habitat.</p> <p>Results</p> <p>In this work, we performed a whole-genome microarray analysis of the <it>X. fastidiosa </it>nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the <it>rpoN </it>null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ⁵⁴-dependent manner. A more complete picture of the σ⁵⁴regulon was achieved by combining the transcriptome data with an <it>in silico </it>search for potential σ⁵⁴-dependent promoters, using a position weight matrix approach. One of these σ⁵⁴-predicted binding sites, located upstream of the <it>glnA </it>gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ⁵⁴-dependent promoter.</p> <p>Conclusions</p> <p>Together, these results show that nitrogen starvation causes intense changes in the <it>X. fastidiosa </it>transcriptome and some of these differentially expressed genes belong to the σ⁵⁴regulon.</p

Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

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

An SOS-regulated operon involved in damage-inducible mutagenesis in Caulobacter crescentus

DNA polymerases of the Y-family, such as Escherichia coli UmuC and DinB, are specialized enzymes induced by the SOS response, which bypass lesions allowing the continuation of DNA replication. umuDC orthologs are absent in Caulobacter crescentus and other bacteria, raising the question about the existence of SOS mutagenesis in these organisms. Here, we report that the C.crescentus dinB ortholog is not involved in damage-induced mutagenesis. However, an operon composed of two hypothetical genes and dnaE2, encoding a second copy of the catalytic subunit of Pol III, is damage inducible in a recA-dependent manner, and is responsible for most ultraviolet (UV) and mitomycin C-induced mutations in C.crescentus. The results demonstrate that the three genes are required for the error-prone processing of DNA lesions. The two hypothetical genes were named imuA and imuB, after inducible mutagenesis. ImuB is similar to proteins of the Y-family of polymerases, and possibly cooperates with DnaE2 in lesion bypass. The mutations arising as a consequence of the activity of the imuAB dnaE2 operon are rather unusual for UV irradiation, including G:C to C:G transversions

Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

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

Global transcriptional response of Caulobacter crescentus to iron availability

Abstract\ud \ud \ud \ud Background\ud 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.\ud \ud \ud \ud Results\ud 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 β-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.\ud \ud \ud \ud Conclusions\ud 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.We are grateful to Michael T. Laub for making the C. crescentus DNA microarray slides available and Carla Rosenberg lab for assistance with the microarray scanning. This work was supported by grant 470663/2011-1 from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). During the course of this work, JFSN and RFL were supported by postdoctoral fellowships, grants 2007/56306-0 and 2008/52874-6, from São Paulo Research Foundation (FAPESP). MVM is partly supported by CNPq.We are grateful to Michael T. Laub for making the C. crescentus DNA microarray slides available and Carla Rosenberg lab for assistance with the microarray scanning. This work was supported by grant 470663/20111 from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). During the course of this work, JFSN and RFL were supported by postdoctoral fellowships, grants 2007/563060 and 2008/528746, from São Paulo Research Foundation (FAPESP). MVM is partly supported by CNPq

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

Abstract\ud \ud Background\ud 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.\ud \ud \ud Results\ud 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.\ud \ud \ud Conclusions\ud 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.FAPESPCNP

O Papel do ensino do português como língua estrangeira na defesa do multicultarismo

As política actuais existentes a nível oficial para a implementação e defesa do ensino da Língua Portuguesa como Língua Estrangeira (L. E.) na Europa e no resto do mundo levam-nos a pensar que são, sobretudo, os casos isolados de leitores portugueses pioneiros, inspirados e marginais que na sua missão individual e afastada lutam pela implementação e defesa desta língua nos seus países de acolhimento. Segundo Volfgram, “cabe ensinar a alguns que o multiculturalismo não está apenas na teoria e sim ao nosso redor, nos elevando realmente à condição de seres humanos” (2005), e o mesmo é dizer que o multiculturalismo começa nas suas bases pela aprendizagem desinteressada e não interesseira das crianças na sua mais tenra idade. Não é impunemente que em países multiculturais como a Bélgica, a Língua Portuguesa ensinada como segunda língua ou como língua estrangeira desempenha um papel preponderante na defesa e na preservação do Português e, em simultâneo, pugna pela defesa incontestável da necessidade incontornável que o multiculturalismo é hoje. É indubitável que a luta contra a xenofobia, a luta pela tolerância e o respeito mútuo, bem como o diálogo profícuo biunívoco não podem sobreviver actualmente sem uma consciencialização da importância das línguas minoritárias, da crioulização, da relação com as línguas maioritárias e da conquista da defesa do multiculturalismo hic et nunc. Abordando algumas opiniões avisadas, esperamos trazer à discussão temas importantes, tais como, a necessidade de articulação de políticas de difusão da língua portuguesa na Europa e no Mundo concertadamente com o Brasil e outros Países Lusófonos, a necessidade de implementação de medidas concretas no terreno para defesa da Língua de Camões fora de Portugal, a sobrevivência do Português que embora sendo minoritária na Europa é uma das línguas mais faladas no mundo, a necessidade da consciencialização para a crescente importância geo-estratégica do Português paralelamente com o recrudescimento do multiculturalismo à escala global

Iron Deficiency Generates Oxidative Stress and Activation of the SOS Response in Caulobacter crescentus

In C. crescentus, iron metabolism is mainly controlled by the transcription factor Fur (ferric uptake regulator). Iron-bound Fur represses genes related to iron uptake and can directly activate the expression of genes for iron-containing proteins. In this work, we used total RNA sequencing (RNA-seq) of wild type C. crescentus growing in minimal medium under iron limitation and a fur mutant strain to expand the known Fur regulon, and to identify novel iron-regulated genes. The RNA-seq of cultures treated with the iron chelator 2-2-dypiridyl (DP) allowed identifying 256 upregulated genes and 236 downregulated genes, being 176 and 204 newly identified, respectively. Sixteen transcription factors and seven sRNAs were upregulated in iron limitation, suggesting that the response to low iron triggers a complex regulatory network. Notably, lexA along with most of its target genes were upregulated, suggesting that DP treatment caused DNA damage, and the SOS DNA repair response was activated in a RecA-dependent manner, as confirmed by RT-qPCR. Fluorescence microscopy assays using an oxidation-sensitive dye showed that wild type cells in iron limitation and the fur mutant were under endogenous oxidative stress, and a direct measurement of cellular H2O2 showed that cells in iron-limited media present a higher amount of endogenous H2O2. A mutagenesis assay using the rpoB gene as a reporter showed that iron limitation led to an increase in the mutagenesis rate. These results showed that iron deficiency causes C. crescentus cells to suffer oxidative stress and to activate the SOS response, indicating an increase in DNA damage

The Transcription Termination Factor Rho Is Essential and Autoregulated in Caulobacter crescentus

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