ABSTRACT
Bacteria adapt to shifts from rapid to slow growth, and have developed strategies for long-term survival during prolonged starvation and stress conditions. We report the regulatory response of C. crescentus to carbon starvation, based on combined high-throughput proteome and transcriptome analyses. Our results identify cell cycle changes in gene expression in response to carbon starvation that involve the prominent role of the FixK FNR/CAP family transcription factor and the CtrA cell cycle regulator. Notably, the SigT ECF sigma factor mediates the carbon starvation-induced degradation of CtrA, while activating a core set of general starvation-stress genes that respond to carbon starvation, osmotic stress, and exposure to heavy metals. Comparison of the response of swarmer cells and stalked cells to carbon starvation revealed four groups of genes that exhibit different expression profiles. Also, cell pole morphogenesis and initiation of chromosome replication normally occurring at the swarmer-to-stalked cell transition are uncoupled in carbon-starved cells.
Subject(s)
Carbon/deficiency , Caulobacter crescentus/genetics , Gene Expression Regulation, Bacterial , Adaptation, Physiological/drug effects , Adaptation, Physiological/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbon/pharmacology , Caulobacter crescentus/cytology , Caulobacter crescentus/drug effects , Gene Expression Profiling , Gene Expression Regulation, Bacterial/drug effects , Gene Regulatory Networks/genetics , Movement/drug effects , Proteome/genetics , Proteome/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Regulon/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Transcription Factors/metabolismABSTRACT
Small noncoding regulatory RNAs (sRNAs) play a key role in the posttranscriptional regulation of many bacterial genes. The genome of Caulobacter crescentus encodes at least 31 sRNAs, and 27 of these sRNAs are of unknown function. An overexpression screen for sRNA-induced growth inhibition along with sequence conservation in a related Caulobacter species led to the identification of a novel sRNA, CrfA, that is specifically induced upon carbon starvation. Twenty-seven genes were found to be strongly activated by CrfA accumulation. One-third of these target genes encode putative TonB-dependent receptors, suggesting CrfA plays a role in the surface modification of C. crescentus, facilitating the uptake of nutrients during periods of carbon starvation. The mechanism of CrfA-mediated gene activation was investigated for one of the genes predicted to encode a TonB-dependent receptor, CC3461. CrfA functions to stabilize the CC3461 transcript. Complementarity between a region of CrfA and the terminal region of the CC3461 5'-untranslated region (5'-UTR) and also the behavior of a deletion of this region and a site-specific base substitution and a 3-base deletion in the CrfA complementary sequence suggest that CrfA binds to a stem-loop structure upstream of the CC3461 Shine-Dalgarno sequence and stabilizes the transcript.
Subject(s)
Caulobacter crescentus/metabolism , RNA, Bacterial/metabolism , RNA, Untranslated/metabolism , 5' Untranslated Regions/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Blotting, Northern , Carbon/metabolism , Caulobacter crescentus/genetics , Gene Expression Regulation, Bacterial/genetics , Gene Expression Regulation, Bacterial/physiology , Molecular Sequence Data , Nucleic Acid Amplification Techniques , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Promoter Regions, Genetic/genetics , RNA, Bacterial/genetics , RNA, Untranslated/genetics , Sequence Homology, Amino AcidABSTRACT
With the aim of identifying genes involved in development and parasite adaptation in cestodes, four coding sequences were isolated from the cyclophyllidean Mesocestoides corti larval stage (tetrathyridium). Genes showed significant similarity to the cysteine-rich secreted protein (CRISP) encoding genes, a large family that includes stage and tissue-specific genes from diverse organisms, many associated with crucial biological processes. The full-length McCrisp2 cDNA encodes a predicted protein of 202 residues in length, containing 10 cysteines and a putative signal peptide. The expression level of McCrisp2 was estimated by Real-time PCR, relative to GAPDH, showing an increase of 75% in segmented worms compared to tetrathyridia. By in situ hybridization, McCrisp2 expression was localized mainly at the larvae apical region of tetrathyridia and in the proglottids of segmented worms. Taken together our results suggest a possible role for M. corti CRISP proteins as ES products, potentially involved in differentiation processes as proposed for homologs in other organisms.
Subject(s)
Glycoproteins/genetics , Helminth Proteins/genetics , Mesocestoides/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/chemistry , Gene Expression , Genes, Homeobox , Glycoproteins/chemistry , Glycoproteins/metabolism , Helminth Proteins/chemistry , Helminth Proteins/metabolism , In Situ Hybridization , Mesocestoides/chemistry , Mice , Molecular Sequence Data , Polymerase Chain Reaction/methods , RNA, Helminth/genetics , RNA, Messenger/genetics , Rats , Sequence AlignmentABSTRACT
To understand the molecular processes regulating morphological changes during cestode life histories we focused on homeodomain (HD) proteins, a family of transcription factors essential for pattern formation during development. In this study we report the isolation of the partial sequence of MvLim, a LIM-HD gene of Mesocestoides corti. Other members of this gene family, characterized in Drosophila melanogaster, Caenorhabditis elegans and vertebrates contribute to cell fate determination of various neuronal subtypes. Phylogenetic analyses showed that MvLim clusters with members of the LIN-11 group and that platyhelminths have at least two different LIM-HD genes. By real time PCR we determined that MvLim expression is 20-fold greater in segmented worms than in tetrathyridia. The enhancement of MvLim expression during strobilation could be associated to changes in the innervation pattern occurring in proglottids development.
Subject(s)
DNA, Helminth/chemistry , Genes, Homeobox/genetics , Homeodomain Proteins/genetics , Mesocestoides/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Gene Expression Regulation, Developmental , Homeodomain Proteins/chemistry , Mesocestoides/classification , Mesocestoides/growth & development , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Sequence Alignment , Up-RegulationABSTRACT
An extremely simple, inexpensive, and safe method is presented, which emulates nucleic acids isolation and electrophoretic analysis as performed in a research environment, in the context of a secondary school hands-on activity. The protocol is amenable to an interdisciplinary approach, taking into consideration the electrical and chemical parameters of the electrophoretic system. Furthermore, the laboratory is framed in a more comprehensive pedagogical setting, which addresses the methodological aspects of a pivotal scientific enterprise such as the Human Genome Project. In this setting, the hands-on activity is complemented with animations, paper models, and discussions. Additionally, our results indicate that the use of borate buffer and agar-agar gels suits many of the experiments included in college-level laboratory activities, which currently make use of more expensive agarose gels and TBE or TAE buffers.
