The cold shock protein (CSP) family includes small polypeptides that are induced upon temperature downshift and stationary phase. The genome of the alphaproteobacterium Caulobacter crescentus encodes four CSPs, with two being induced by cold shock and two at the onset of stationary phase. In order to identify the environmental signals and cell factors that are involved in cspD expression at stationary phase, we have analyzed cspD transcription during growth under several nutrient conditions. The results showed that expression of cspD was affected by the medium composition and was inversely proportional to the growth rate. The maximum levels of expression were decreased in a spoT mutant, indicating that ppGpp may be involved in the signalization for carbon starvation induction of cspD. A Tn5 mutant library was screened for mutants with reduced cspD expression, and 10 clones that showed at least a 50% reduction in expression were identified. Among these, a strain with a transposon insertion into a response regulator of a two-component system showed no induction of cspD at stationary phase. This protein (SpdR) was able to acquire a phosphate group from its cognate histidine kinase, and gel mobility shift assay and DNase I footprinting experiments showed that it binds to an inverted repeat sequence of the cspD regulatory region. A mutated SpdR with a substitution of the conserved aspartyl residue that is the probable phosphorylation site is unable to bind to the cspD regulatory region and to complement the spdR mutant phenotype.

Siderophore nutrition tests with strain NA1000 revealed that it utilized a variety of ferric hydroxamate siderophores, including asperchromes, ferrichromes, ferrichrome A, malonichrome, and ferric aerobactin, as well as hemin and hemoglobin. did not transport ferrioxamine B or ferric catecholates. Because it did not use ferric enterobactin, the catecholate aposiderophore was an effective agent for iron deprivation. We determined the kinetics and thermodynamics of [Fe]apoferrichrome and Fe-citrate binding and transport by NA1000. Its affinity and uptake rate for ferrichrome (equilibrium dissociation constant [ ], 1 nM; Michaelis-Menten constant [ ], 0.1 nM; , 19 pMol/10 cells/min) were similar to those of FhuA. Transport properties forFe-citrate were similar to those of FecA ( , 5.3 nM; , 29 pMol/10 cells/min). Bioinformatic analyses implicated Fur-regulated loci ,, , and as TonB-dependent transporters (TBDT) that participate in iron acquisition. We resolved TBDT with elevated expression under high- or low-iron conditions by SDS-PAGE of sodium sarcosinate cell envelope extracts, excised bands of interest, and analyzed them by mass spectrometry. These data identified five TBDT: three were overexpressed during iron deficiency (00028, 02277, and 03023), and 2 were overexpressed during iron repletion (00210 and 01196). CLUSTALW analyses revealed homology of putative TBDT 02277 to FepA and BtuB. A Δ mutant did not transport hemin or hemoglobin in nutrition tests, leading us to designate the structural gene as (for eme/hemoglobintilization). The physiological roles of the 62 putative TBDT of are mostly unknown, as are their evolutionary relationships to TBDT of other bacteria. We biochemically studied the iron uptake systems of , identified potential iron transporters, and clarified the phylogenetic relationships among its numerous TBDT. Our findings identified the first outer membrane protein involved in iron acquisition by, its heme/hemoglobin transporter (HutA).

The cold shock response in bacteria involves the expression of low-molecular weight cold shock proteins (CSPs) containing a nucleic acid-binding cold shock domain (CSD), which are known to destabilize secondary structures on mRNAs, facilitating translation at low temperatures. Caulobacter crescentus cspA and cspB are induced upon cold shock, while cspC and cspD are induced during stationary phase. In this work, we determined a new coding sequence for the cspC gene, revealing that it encodes a protein containing two CSDs. The phenotypes of C. crescentus csp mutants were analyzed, and we found that cspC is important for cells to maintain viability during extended periods in stationary phase. Also, cspC and cspCD strains presented altered morphology, with frequent non-viable filamentous cells, and cspCD also showed a pronounced cell death at late stationary phase. In contrast, the cspAB mutant presented increased viability in this phase, which is accompanied by an altered expression of both cspC and cspD, but the triple cspABD mutant loses this characteristic. Taken together, our results suggest that there is a hierarchy of importance among the csp genes regarding stationary phase viability, which is probably achieved by a fine tune balance of the levels of these proteins.

BackgroundThe 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.ResultsThe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1845-1) contains supplementary material, which is available to authorized users.

À Profa. Dra. Marilis do Valle Marques pela orientação zelosa, pela confiança e, sobretudo, pelo profissionalismo e dedicação a seus alunos. Aos colegas de laboratório André, Carolina, José, Ricardo, Valéria Italiani, Valéria Karla, Vânia e Ynés, pelo bom ambiente de trabalho, pela colaboração e principalmente pela amizade. Aos ex-colegas de laboratório Émerson, João, Letícia, Lígia, Rafael e Zuleta, e às colegas "ainda não oficiais" Juliana e Mirian, pelos mesmos motivos. À Elza, por fornecer as bases para este trabalho e pelos ensinamentos valiosos desde minha Iniciação Científica, além da amizade. Aos componentes da banca de qualificação pelas sugestões. Aos professores, funcionários e colegas do Departamento de Microbiologia. Às funcionárias Íris e Marlene pela eficiência, apoio técnico e amizade. À Profa. Dra. Regina Lúcia Baldini e a Ana Laura pela ajuda prestada na obtenção de anticorpos. Ao técnico Carlos, do biotério do Instituto de Ciências Biomédicas da USP, pela ajuda na imunização de coelhos. À FAPESP pelo auxílio financeiro. Às secretárias Alice, Ana e Naíde pela ajuda sempre que foram solicitadas. Às amigas e colegas de laboratório Carol e Val Karla, pelas conversas, pelos momentos divertidos e por toda a ajuda que me ofereceram, não só no laboratório como fora dele. À minha mãe, Rosa Maria, pelo apoio, compreensão, amor e pelos valores transmitidos a mim. Ao meu querido namorado Sergio, pela cumplicidade, pela compreensão, pelo afeto, pela empatia e por tornar meus dias mais felizes a seu lado. RESUMO BALHESTEROS, H. Análise do papel do gene cspC de Caulobacter crescentus e de sua regulação. 2009. 126 f. Dissertação (Mestrado