The Leptospira interrogans proteome's response to zinc highlights the potential involvement of this metal in translational-machinery and virulence.

Leptospires, as motile Gram-negative bacteria, employ sophisticated strategies for efficient invasion and dissemination within their hosts. In response, hosts counteract pathogens through nutritional immunity, a concept involving the deprivation of essential metals such as zinc. Zinc, pivotal in modulating pathogen-host interactions, influences proteins structural, catalytic, and regulatory functions. A comprehensive understanding of how leptospires regulate intracellular zinc availability is crucial for deciphering their survival mechanisms. This study explores the proteomic profile of Leptospira interrogans sv. Copenhageni str. 10A cultivated in Ellinghausen-McCullough-Johnson-Harris medium supplemented with the zinc chelator TPA or ZnCl2. Among the 2161 proteins identified, 488 were subjected to scrutiny, revealing 102 less abundant and 81 more abundant in response to TPA. Of these 488 proteins, 164 were exclusive to the presence of TPA and 141 were exclusive to the zinc-enriched conditions. Differentially expressed proteins were classified into clusters of orthologous groups (COGs) with a distribution in metabolic functions (37.8%), information storage/processing (21.08%), cellular processes/signaling (28.04%), and poorly characterized proteins (10.65%). Differentially expressed proteins are putatively involved in processes like 1-carbon compound metabolism, folate biosynthesis, and amino acid/nucleotide synthesis. Zinc availability significantly impacted key processes putatively related to leptospires' interactions with their host, such as motility, biofilm formation, and immune escape. Under conditions of higher zinc concentration, ribosomal proteins, chaperones and components of transport systems were observed, highlighting interactions between regulatory networks responsive to zinc and iron in L. interrogans. This study not only revealed hypothetical proteins potentially related to zinc homeostasis, but also identified possible virulence mechanisms and pathogen-host adaptation strategies influenced by the availability of this metal. There is an urgent need, based on these data, for further in-depth studies aimed at detailing the role of zinc in these pathways and mechanisms, which may ultimately determine more effective therapeutic approaches to combat Leptospira infections.

The DEAD-box RNA helicase RhlB is required for efficient RNA processing at low temperature in Caulobacter.

IMPORTANCE: One of the most important control points in gene regulation is RNA stability, which determines the half-life of a transcript from its transcription until its degradation. Bacteria have evolved a sophisticated multi-enzymatic complex, the RNA degradosome, which is dedicated mostly to RNA turnover. The combined activity of RNase E and the other RNA degradosome enzymes provides an efficient pipeline for the complete degradation of RNAs. The DEAD-box RNA helicases are very often found in RNA degradosomes from phylogenetically distant bacteria, confirming their importance in unwinding structured RNA for subsequent degradation. This work showed that the absence of the RNA helicase RhlB in the free-living Alphaproteobacterium Caulobacter crescentus causes important changes in gene expression and cell physiology. These are probably due, at least in part, to inefficient RNA processing by the RNA degradosome, particularly at low-temperature conditions.

Fluorescent sensors of siderophores produced by bacterial pathogens.

Siderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with ∼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria.

Cold Regulation of Genes Encoding Ion Transport Systems in the Oligotrophic Bacterium Caulobacter crescentus.

In this study, we characterize the response of the free-living oligotrophic alphaproteobacterium Caulobacter crescentus to low temperatures by global transcriptomic analysis. Our results showed that 656 genes were upregulated and 619 were downregulated at least 2-fold after a temperature downshift. The identified differentially expressed genes (DEG) belong to several functional categories, notably inorganic ion transport and metabolism, and a subset of these genes had their expression confirmed by reverse transcription quantitative real-time PCR (RT-qPCR). Several genes belonging to the ferric uptake regulator (Fur) regulon were downregulated, indicating that iron homeostasis is relevant for adaptation to cold. Several upregulated genes encode proteins that interact with nucleic acids, particularly RNA: cspA, cspB, and the DEAD box RNA helicases rhlE, dbpA, and rhlB. Moreover, 31 small regulatory RNAs (sRNAs), including the cell cycle-regulated noncoding RNA (ncRNA) CcnA, were upregulated, indicating that posttranscriptional regulation is important for the cold stress response. Interestingly, several genes related to transport were upregulated under cold stress, including three AcrB-like cation/multidrug efflux pumps, the nitrate/nitrite transport system, and the potassium transport genes kdpFABC. Further characterization showed that kdpA is upregulated in a potassium-limited medium and at a low temperature in a SigT-independent way. kdpA mRNA is less stable in rho and rhlE mutant strains, but while the expression is positively regulated by RhlE, it is negatively regulated by Rho. A kdpA-deleted strain was generated, and its viability in response to osmotic, acidic, or cold stresses was determined. The implications of such variation in the gene expression for cold adaptation are discussed. IMPORTANCE Low-temperature stress is an important factor for nucleic acid stability and must be circumvented in order to maintain the basic cell processes, such as transcription and translation. The oligotrophic lifestyle presents further challenges to ensure the proper nutrient uptake and osmotic balance in an environment of slow nutrient flow. Here, we show that in Caulobacter crescentus, the expression of the genes involved in cation transport and homeostasis is altered in response to cold, which could lead to a decrease in iron uptake and an increase in nitrogen and high-affinity potassium transport by the Kdp system. This previously uncharacterized regulation of the Kdp transporter has revealed a new mechanism for adaptation to low temperatures that may be relevant for oligotrophic bacteria.

Protein Pulldown Assays to Monitor the Composition of the Bacterial RNA Degradosome.

The method of co-immunoprecipitation (co-IP or pulldown) enables the identification of proteins interacting in macromolecular assemblies, through the purification of a key protein by affinity chromatography using specific antibodies immobilized on a matrix. The advantages of using epitope-tagged proteins include the ability to use commercially available antibodies for affinity purifications, and typically they do not disrupt the structure of the protein complexes. Here we describe the utilization of an epitope-tagged version of Caulobacter crescentus RNase E in order to determine the composition of the RNA degradosome under different growth conditions. Several proteins that interact with the RNA degradosome were identified.

Identification of Hfq-binding RNAs in Caulobacter crescentus.

Small RNAs are important for post-transcriptional regulation of gene expression, affecting stability and activity of their target mRNAs. The bacterial Sm-like protein Hfq is required to promote pairing between both RNAs when their sequence complementarity is limited. To provide a first global view on the post-transcriptional landscape of the α-proteobacterium Caulobacter crescentus, we have identified the Hfq-binding RNAs employing High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). A total of 261 RNAs, including 3 unannotated RNAs, were successfully identified and classified according to putative function. Moreover, possible interactions between the identified sRNAs with mRNA targets were postulated through computational target predictions.

OxyR and the hydrogen peroxide stress response in Caulobacter crescentus.

Oxidative stress generated by hydrogen peroxide is faced by bacteria when encountering hostile environments. In order to define the physiological and regulatory networks controlling the oxidative stress response in the free-living bacterium Caulobacter crescentus, a whole transcriptome analysis of wild type and ΔoxyR strains in the presence of hydrogen peroxide for two different exposure times was carried out. The C. crescentus response to H2O2 includes a decrease of the assimilative sulfate reduction and a shift in the amino acid synthesis pathways into favoring the synthesis of histidine. Moreover, the expression of genes encoding enzymes for the depolymerization of polyhydroxybutyrate was increased, and the RpoH-dependent genes were severely repressed. Based on the expression pattern and sequence analysis, we postulate that OxyR is probably directly required for the induction of three genes (katG, ahpCF). The putative binding of OxyR to the ahpC regulatory region could be responsible for the use of one of two alternative promoters in response to oxidative stress. Nevertheless, OxyR is required for the expression of 103 genes in response to H2O2. Fur and part of its regulon were differentially expressed in response to hydrogen peroxide independently of OxyR. The non-coding RNA OsrA was upregulated in both strains, and an in silico analysis indicated that it may have a regulatory role. This work characterizes the physiological response to H2O2 in C. crescentus, the regulatory networks and differentially regulated genes in oxidative stress and the participation of OxyR in this process. It is proposed that besides OxyR, a second layer of regulation may be achieved by a small regulatory RNA and other transcriptional regulators.

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.

Effect of SOS-induced levels of imuABC on spontaneous and damage-induced mutagenesis in Caulobacter crescentus.

imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli.

Molecular characterization of Caulobacter crescentus mutator strains.

Mutator strains were identified by screening random Tn5 insertion clones of Caulobacter crescentus. We identified clones with robust increases in mutation rates with Tn5 insertions in the mutY, mutS, mutL and uvrD genes, known to act in mutation-preventing pathways in Escherichia coli. Analysis of mutations in the rpoB gene revealed that in both the parental strain and mismatch repair-deficient mutants, A:T→G:C transitions predominate by a large margin over C:G→T:A. We have also investigated the role of the error-prone polymerase encoded by imuC (dnaE2) in spontaneous mutagenesis, and found that a imuC mutant strain shows mutation rates and sequences comparable to the parental strain. Our study characterizes for the first time mutator strains in a member of the alphaproteobacteria group. In spite of the limitations of using a single marker, possible reasons for the observed mutational bias are discussed in the light of the repertoire of DNA repair genes in this bacterium.

Association of the Cold Shock DEAD-Box RNA Helicase RhlE to the RNA Degradosome in Caulobacter crescentus.

In diverse bacterial lineages, multienzyme assemblies have evolved that are central elements of RNA metabolism and RNA-mediated regulation. The aquatic Gram-negative bacterium Caulobacter crescentus, which has been a model system for studying the bacterial cell cycle, has an RNA degradosome assembly that is formed by the endoribonuclease RNase E and includes the DEAD-box RNA helicase RhlB. Immunoprecipitations of extracts from cells expressing an epitope-tagged RNase E reveal that RhlE, another member of the DEAD-box helicase family, associates with the degradosome at temperatures below those optimum for growth. Phenotype analyses of rhlE, rhlB, and rhlE rhlB mutant strains show that RhlE is important for cell fitness at low temperature and its role may not be substituted by RhlB. Transcriptional and translational fusions of rhlE to the lacZ reporter gene and immunoblot analysis of an epitope-tagged RhlE indicate that its expression is induced upon temperature decrease, mainly through posttranscriptional regulation. RNase E pulldown assays show that other proteins, including the transcription termination factor Rho, a second DEAD-box RNA helicase, and ribosomal protein S1, also associate with the degradosome at low temperature. The results suggest that the RNA degradosome assembly can be remodeled with environmental change to alter its repertoire of helicases and other accessory proteins.IMPORTANCE DEAD-box RNA helicases are often present in the RNA degradosome complex, helping unwind secondary structures to facilitate degradation. Caulobacter crescentus is an interesting organism to investigate degradosome remodeling with change in temperature, because it thrives in freshwater bodies and withstands low temperature. In this study, we show that at low temperature, the cold-induced DEAD-box RNA helicase RhlE is recruited to the RNA degradosome, along with other helicases and the Rho protein. RhlE is essential for bacterial fitness at low temperature, and its function may not be complemented by RhlB, although RhlE is able to complement for rhlB loss. These results suggest that RhlE has a specific role in the degradosome at low temperature, potentially improving adaptation to this condition.

TonB-Dependent Heme/Hemoglobin Utilization by Caulobacter crescentus HutA.

Siderophore nutrition tests with Caulobacter crescentus 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. C. crescentus 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 [59Fe]apoferrichrome and 59Fe-citrate binding and transport by NA1000. Its affinity and uptake rate for ferrichrome (equilibrium dissociation constant [Kd ], 1 nM; Michaelis-Menten constant [KM ], 0.1 nM; Vmax, 19 pMol/109 cells/min) were similar to those of Escherichia coli FhuA. Transport properties for 59Fe-citrate were similar to those of E. coli FecA (KM , 5.3 nM; Vmax, 29 pMol/109 cells/min). Bioinformatic analyses implicated Fur-regulated loci 00028, 00138, 02277, and 03023 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 Escherichia coli FepA and BtuB. A Δ02277 mutant did not transport hemin or hemoglobin in nutrition tests, leading us to designate the 02277 structural gene as hutA (for heme/hemoglobin utilization).IMPORTANCE The physiological roles of the 62 putative TBDT of C. crescentus are mostly unknown, as are their evolutionary relationships to TBDT of other bacteria. We biochemically studied the iron uptake systems of C. crescentus, 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 C. crescentus, its heme/hemoglobin transporter (HutA).

Role and regulation of ferritin-like proteins in iron homeostasis and oxidative stress survival of Caulobacter crescentus.

Iron is an essential nutrient that is poorly available to living organisms but can be harmful when in excess due to the production of reactive oxygen species. Bacteria and other organisms use iron storage proteins called ferritins to avoid iron toxicity and as a safe iron source in the cytosol. The alpha-proteobacterium Caulobacter crescentus has two putative ferritins, Bfr and Dps, and some other proteins belonging to the ferritin-like superfamily, among them the one encoded by CC_0557. In this work, we have analyzed the role and regulation of these three putative ferritin-like proteins. Using lacZ-transcriptional fusions, we found that bfr expression is positively regulated (2.5-fold induction) by the Fe-responsive regulator Fur in iron sufficiency, as expected for an iron storage protein. Expression of dps was induced 1.5-fold in iron limitation in a Fur-independent manner, while the expression of the product of CC_0557 was unaffected by either iron supply or Fur. With respect to growth phase, while bfr expression was constant during growth, expression of dps (1.4-fold) and CC_0557 (around seven times) increased in the transition from exponential to stationary phase. Deletion mutant strains for each gene and a double dps/bfr mutant were obtained and tested for oxidative stress resistance. The dps mutant was very sensitive to H2O2, and this phenotype was not relieved by the addition of the iron chelator 2',2-dipyridyl in the conditions tested. While bfr and CC_0557 showed no phenotype as to H2O2 resistance, the double dps/bfr mutant had a similar phenotype to the dps mutation alone. These findings indicate that in C. crescentus Bfr contributes to iron homeostasis and Dps has a role in protection against oxidative stress. The role of the protein CC_0557 containing a ferritin-like fold remains unclear.

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.

Analysis of the Caulobacter crescentus Zur regulon reveals novel insights in zinc acquisition by TonB-dependent outer membrane proteins.

BACKGROUND: Intracellular zinc concentration needs to be maintained within strict limits due to its toxicity at high levels, and this is achieved by a finely regulated balance between uptake and efflux. Many bacteria use the Zinc Uptake Regulator Zur to orchestrate zinc homeostasis, but little is known regarding the transport of this metal across the bacterial outer membrane. RESULTS: In this work we determined the Caulobacter crescentus Zur regulon by global transcriptional and in silico analyses. Among the genes directly repressed by Zur in response to zinc availability are those encoding a putative high affinity ABC uptake system (znuGHI), three TonB-dependent receptors (znuK, znuL and znuM) and one new putative transporter of a family not yet characterized (zrpW). Zur is also directly involved in the activation of a RND and a P-type ATPase efflux systems, as revealed by ß-galactosidase and site-directed mutagenesis assays. Several genes belonging to the Fur regulon were also downregulated in the zur mutant, suggesting a putative cross-talk between Zur and Fur regulatory networks. Interestingly, a phenotypic analysis of the znuK and znuL mutants has shown that these genes are essential for growth under zinc starvation, suggesting that C. crescentus uses these TonB-dependent outer membrane transporters as key zinc scavenging systems. CONCLUSIONS: The characterization of the C. crescentus Zur regulon showed that this regulator coordinates not only uptake, but also the extrusion of zinc. The uptake of zinc by C. crescentus in conditions of scarcity of this metal is highly dependent on TonB-dependent receptors, and the extrusion is mediated by an RND and P-type ATPase transport systems. The absence of Zur causes a disturbance in the dynamic equilibrium of zinc intracellular concentration, which in turn can interfere with other regulatory networks as seen for the Fur regulon.

Leptospira interrogans serovar copenhageni harbors two lexA genes involved in SOS response.

Bacteria activate a regulatory network in response to the challenges imposed by DNA damage to genetic material, known as the SOS response. This system is regulated by the RecA recombinase and by the transcriptional repressor lexA. Leptospira interrogans is a pathogen capable of surviving in the environment for weeks, being exposed to a great variety of stress agents and yet retaining its ability to infect the host. This study aims to investigate the behavior of L. interrogans serovar Copenhageni after the stress induced by DNA damage. We show that L. interrogans serovar Copenhageni genome contains two genes encoding putative LexA proteins (lexA1 and lexA2) one of them being potentially acquired by lateral gene transfer. Both genes are induced after DNA damage, but the steady state levels of both LexA proteins drop, probably due to auto-proteolytic activity triggered in this condition. In addition, seven other genes were up-regulated following UV-C irradiation, recA, recN, dinP, and four genes encoding hypothetical proteins. This set of genes is potentially regulated by LexA1, as it showed binding to their promoter regions. All these regions contain degenerated sequences in relation to the previously described SOS box, TTTGN 5CAAA. On the other hand, LexA2 was able to bind to the palindrome TTGTAN10TACAA, found in its own promoter region, but not in the others. Therefore, the L. interrogans serovar Copenhageni SOS regulon may be even more complex, as a result of LexA1 and LexA2 binding to divergent motifs. New possibilities for DNA damage response in Leptospira are expected, with potential influence in other biological responses such as virulence.

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.

Two RND proteins involved in heavy metal efflux in Caulobacter crescentus belong to separate clusters within proteobacteria.

BACKGROUND: Heavy metal Resistance-Nodulation-Division (HME-RND) efflux systems help Gram-negative bacteria to keep the intracellular homeostasis under high metal concentrations. These proteins constitute the cytoplasmic membrane channel of the tripartite RND transport systems. Caulobacter crescentus NA1000 possess two HME-RND proteins, and the aim of this work was to determine their involvement in the response to cadmium, zinc, cobalt and nickel, and to analyze the phylogenetic distribution and characteristic signatures of orthologs of these two proteins. RESULTS: Expression assays of the czrCBA operon showed significant induction in the presence of cadmium and zinc, and moderate induction by cobalt and nickel. The nczCBA operon is highly induced in the presence of nickel and cobalt, moderately induced by zinc and not induced by cadmium. Analysis of the resistance phenotype of mutant strains showed that the ΔczrA strain is highly sensitive to cadmium, zinc and cobalt, but resistant to nickel. The ΔnczA strain and the double mutant strain showed reduced growth in the presence of all metals tested. Phylogenetic analysis of the C. crescentus HME-RND proteins showed that CzrA-like proteins, in contrast to those similar to NczA, are almost exclusively found in the Alphaproteobacteria group, and the characteristic protein signatures of each group were highlighted. CONCLUSIONS: The czrCBA efflux system is involved mainly in response to cadmium and zinc with a secondary role in response to cobalt. The nczCBA efflux system is involved mainly in response to nickel and cobalt, with a secondary role in response to cadmium and zinc. CzrA belongs to the HME2 subfamily, which is almost exclusively found in the Alphaproteobacteria group, as shown by phylogenetic analysis. NczA belongs to the HME1 subfamily which is more widespread among diverse Proteobacteria groups. Each of these subfamilies present distinctive amino acid signatures.

Cold shock genes cspA and cspB from Caulobacter crescentus are posttranscriptionally regulated and important for cold adaptation.

Cold shock proteins (CSPs) are nucleic acid binding chaperones, first described as being induced to solve the problem of mRNA stabilization after temperature downshift. Caulobacter crescentus has four CSPs: CspA and CspB, which are cold induced, and CspC and CspD, which are induced only in stationary phase. In this work we have determined that the synthesis of both CspA and CspB reaches the maximum levels early in the acclimation phase. The deletion of cspA causes a decrease in growth at low temperature, whereas the strain with a deletion of cspB has a very subtle and transient cold-related growth phenotype. The cspA cspB double mutant has a slightly more severe phenotype than that of the cspA mutant, suggesting that although CspA may be more important to cold adaptation than CspB, both proteins have a role in this process. Gene expression analyses were carried out using cspA and cspB regulatory fusions to the lacZ reporter gene and showed that both genes are regulated at the transcriptional and posttranscriptional levels. Deletion mapping of the long 5'-untranslated region (5'-UTR) of each gene identified a common region important for cold induction, probably via translation enhancement. In contrast to what was reported for other bacteria, these cold shock genes have no regulatory regions downstream from ATG that are important for cold induction. This work shows that the importance of CspA and CspB to C. crescentus cold adaptation, mechanisms of regulation, and pattern of expression during the acclimation phase apparently differs in many aspects from what has been described so far for other bacteria.