Gotowa bibliografia na temat „Carbon Starvation Gene yjiY”

ABSTRACTThis paper describes the transcriptional adaptations of nongrowing, retentostat cultures ofLactococcus lactisto starvation. Near-zero-growth cultures (μ = 0.0001 h−1) obtained by extended retentostat cultivation were exposed to starvation by termination of the medium supply for 24 h, followed by a recovery period of another 24 h by reinitiating the medium supply to the retentostat culture. During starvation, the viability of the culture was largely retained, and the expression of genes involved in transcription and translational machineries, cell division, and cell membrane energy metabolism was strongly repressed. Expression of these genes was largely recovered following the reinitiation of the medium supply. Starvation triggered the elevated expression of genes associated with synthesis of branched-chain amino acids, histidine, purine, and riboflavin. The expression of these biosynthesis genes was found to remain at an elevated level after reinitiation of the medium supply. In addition, starvation induced the complete gene set predicted to be involved in natural competence inL. lactisKF147, and the elevated expression of these genes was sustained during the subsequent recovery period, but our attempts to experimentally demonstrate natural transformation in these cells failed. Mining the starvation response gene set identified a conservedcis-acting element that resembles the lactococcal CodY motif in the upstream regions of genes associated with transcription and translational machineries, purine biosynthesis, and natural transformation inL. lactis, suggesting a role for CodY in the observed transcriptome adaptations to starvation in nongrowing cells.

An increasingly apparent role of noncoding RNA (ncRNAs) is to coordinate gene expression during environmental stress. A mounting body of evidence implicates small RNAs (sRNAs) as key drivers of Salmonella stress survival. Generally thought to be 50–500 nucleotides in length and to occur in intergenic regions, sRNAs typically regulate protein expression through base pairing with mRNA targets. In this work, through employing a refined definition of sRNAs allowing for shorter sequences and sRNA loci to overlap with annotated protein-coding gene loci, we have identified 475 previously unannotated sRNAs that are significantly differentially expressed during carbon starvation (C-starvation). Northern blotting and quantitative RT-PCRs confirm the expressions and identities of several of these novel sRNAs, and our computational analyses find the majority to be highly conserved and structurally related to known sRNAs. Importantly, we show that deletion of one of the sRNAs dynamically expressed during C-starvation, sRNA4130247, significantly impairs the Salmonella C-starvation response (CSR), confirming its involvement in the Salmonella CSR. In conclusion, the work presented here provides the first-ever characterization of intragenic sRNAs in Salmonella, experimentally confirms that sRNAs dynamically expressed during the CSR are directly involved in stress survival, and more than doubles the Salmonella enterica sRNAs described to date.

Microorganisms adopt diverse mechanisms to adapt to fluctuations of nutrients. Glucose is the preferred carbon and energy source for yeast. Yeast cells have developed many strategies to protect themselves from the negative impact of glucose starvation. Studies have indicated a significant increase of carotenoids in red yeast under glucose starvation. However, their regulatory mechanism is still unclear. In this study, we investigated the regulatory mechanism of carotenoid biosynthesis in Rhodosporidium kratochvilovae YM25235 under glucose starvation. More intracellular reactive oxygen species (ROS) was produced when glucose was exhausted. Enzymatic and non-enzymatic (mainly carotenoids) antioxidant systems in YM25235 were induced to protect cells from ROS-related damage. Transcriptome analysis revealed massive gene expression rearrangement in YM25235 under glucose starvation, leading to alterations in alternative carbon metabolic pathways. Some potential pathways for acetyl-CoA and then carotenoid biosynthesis, including fatty acid β-oxidation, amino acid metabolism, and pyruvate metabolism, were significantly enriched in KEGG analysis. Overexpression of the fatty acyl-CoA oxidase gene (RkACOX2), the first key rate-limiting enzyme of peroxisomal fatty acid β-oxidation, demonstrated that fatty acid β-oxidation could increase the acetyl-CoA and carotenoid concentration in YM25235. These findings contribute to a better understanding of the overall response of red yeast to glucose starvation and the regulatory mechanisms governing carotenoid biosynthesis under glucose starvation.

The stability of mRNA was investigated for the first time at the genomic scale during carbon starvation adaptation of Lactococcus lactis IL1403. In exponential phase, mRNA half-lives were correlated positively to open reading frame length. A polypurine sequence, AGGAG, was identified as a putative 5′-stabilizer and inverted repeated sequences as a 3′-destabilizer. These original findings suggested that multiple pathways of mRNA degradation should coexist: internal cleavage, endonuclease cleavage initiated at the 5′-end, and exonuclease attack at the 3′-end. During carbon starvation adaptation, mRNA stability globally increased, but specific mechanisms allowing a wide range of stabilization factors between genes and differential kinetic evolution were involved. A formal method allowing the quantification of the relative influences of transcription and degradation on the mRNA pool control was developed and applied in L. lactis. Gene expression was mostly controlled by altered transcription prior to carbon source exhaustion, while the influence of mRNA stability increased during the starvation phase. This study highlighted that stability modulation in response to adverse growth conditions can govern gene regulation to the same extent as transcription in bacteria.

ABSTRACT Transcription of the clpP-clpX operon ofEscherichia coli leads to the production of two different sizes of transcripts. In log phase, the level of the longer transcript is higher than the level of the shorter transcript. Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases. The longer transcript consists of the entireclpP-clpX operon, whereas the shorter transcript contains the entire clpP gene but none of the clpXcoding sequence. The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation. Primer extension experiments suggest that there is increased usage of the ς32-dependent promoter of the clpP-clpXoperon within 15 min after the start of carbon starvation. Expression of the clpP-clpX operon from the promoters upstream of theclpP gene decreases to a very low level by 20 min after the onset of carbon starvation. Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript. The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation. E. coli rpoBmutations that affect transcription termination efficiency alter the ratio of the shorter clpP-clpX transcript to the longer transcript. The E. coli rpoB3595 mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain. In contrast, the rpoB8 mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter clpP-clpXtranscript when E. coli is in log phase. These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript.

To understand the relationship between carbon or nitrogen utilization and iron homeostasis, we performed an iron uptake assay with several deletion mutants with partial defects in carbon or nitrogen metabolism. Among them, some deletion mutants defective in carbon metabolism partially and the MEP2 deletion mutant showed lower iron uptake activity than the wild type. Mep2 is known as a high-affinity ammonia transporter in Saccharomyces cerevisiae. Interestingly, we found that nitrogen starvation resulted in lower iron uptake activity than that of wild-type cells without downregulation of the genes involved in the high-affinity iron uptake system FET3/FTR1. However, the gene expression of FRE1 and CTR1 was downregulated by nitrogen starvation. The protein level of Ctr1 was also decreased by nitrogen starvation, and addition of copper to the nitrogen starvation medium partially restored iron uptake activity. However, the expression of MAC1, which is a copper-responsive transcriptional activator, was not downregulated by nitrogen starvation at the transcriptional level but was highly downregulated at the translational level. Mac1 was downregulated dramatically under nitrogen starvation, and treatment with MG132, which is an inhibitor of proteasome-dependent protein degradation, partially attenuated the downregulation of Mac1. Taken together, these results suggest that nitrogen starvation downregulates the high-affinity iron uptake system by degrading Mac1 in a proteasome-dependent manner and eventually downregulates copper metabolism.

ABSTRACT A gene homologous to the rpoS gene of Escherichia coli was cloned from a Pseudomonas putida KT2440 gene bank by complementation of the rpoS-deficient strainE. coli ZK918. The rpoS gene of P. putida complemented the acid sensitivity and catalase deficiency of the rpoS mutant of E. coli and stimulated expression of the RpoS-controlled promoter,bolAp 1. The gene was sequenced and found to be highly similar to the rpoS genes of other gram-negative bacteria. Like in other gram-negative bacteria, a homolog of thenlpD gene was found upstream to the rpoS gene. A transcriptional fusion of the promoter of the P. putida rpoS gene to the luxAB genes from Vibrio harveyi was constructed and used as an inactivated allele ofrpoS for gene replacement of the wild-type copy in the chromosome of P. putida. The resultantrpoS mutant of P. putida, C1R1, showed reduced survival of carbon starvation and reduced cross-protection against other types of stress in cells starved for carbon, in particular after a challenge with ethanol. Survival in soil amended with m-methylbenzoate was also reduced in the mutant strain P. putida C1R1. The RpoS protein ofP. putida controls the expression of more than 50 peptides, which are normally expressed in cells after a short period of carbon starvation.

Starch provides plants with carbon and energy during stressful periods; however, relatively few regulators of starch metabolism under stress-induced carbon starvation have been discovered. We studied a protein kinase Ser/Thr/Tyr (STY) 46, identified by gene co-expression network analysis as a potential regulator of the starch starvation response in Arabidopsis thaliana. We showed that STY46 was induced by (1) abscisic acid and prolonged darkness, (2) by abiotic stressors, including salinity and osmotic stress, and (3) by conditions associated with carbon starvation. Characterization of STY46 T-DNA knockout mutants indicated that there was functional redundancy among the STY gene family, as these genotypes did not show strong phenotypes. However, Arabidopsis with high levels of STY46 transcripts (OE-25) grew faster at the early seedling stage, had higher photosynthetic rates, and more carbon was stored as protein in the seeds under control conditions. Further, OE-25 source leaf accumulated more sugars under 100 mM NaCl stress, and salinity also accelerated root growth, which is consistent with an adaptive response. Salt-stressed OE-25 partitioned 14C towards sugars and amino acids, and away from starch and protein in source leaves. Together, these findings suggested that STY46 may be part of the salinity stress response pathway that utilizes starch during early plant growth.

Carbon starvation genes mediate the cross-talk between metabolism and pathogenesis of Salmonella Typhimurium Salmonella enterica serovar Typhimurium (S. Typhimurium) can infect a wide range of host animals to cause diseases like gastroenteritis and typhoid fever. Therefore it serves as an ideal model organism to study Salmonella pathogenesis. The adaptability of Salmonella to diverse environmental conditions is due to its ability to utilize a plethora of nutrients. While various carbon and nitrogen sources are supplied by the host, the role of peptides as alternate nutrient source for Salmonella is not clearly defined. These peptides are presented by the host as antimicrobial peptides, but can serve as nutrients too, once taken up by peptide transporters and digested by the pathogen. The importance of peptide transporters is also reported for alternate functions such as quorum sensing, competence, chemotaxis and virulence. The ABC transporters of peptides are well studied in Salmonella, whereas little is known about the putative peptide transporter family named as carbon starvation genes which lack ATP binding site. Two carbon starvation (cst) genes, cstA and yjiY, are the only genes known to belong to this group of peptide transporters in Salmonella. cstA was previously reported to be required for virulence of Salmonella in C. elegans. To establish the role of cst genes in the metabolism and pathogenesis of S. Typhimurium, the knockout strains for the genes cstA and yjiY in S. Typhimurium, denoted as ΔcstA and ΔyjiY, were generated. The metabolic capacity of these mutants was checked by phenotype microarray revealing that cst knockout strains were compromised in peptide metabolism and ΔyjiY strain showed remarkable difference from the wild type in the ability to utilize a few peptides. Fluorescent peptide uptake assay showed reduced uptake of specific dipeptides by ΔyjiY strain. Thus, cst genes contribute to metabolism of Salmonella by transporting specific peptides. Upon infecting C. elegans, ΔcstA was unable to colonize the intestine of the worm verifying the reported role of cstA. However, in mammalian model systems, ΔyjiY, but not ΔcstA, was unable to invade various types of host cells, attributed to defective adhesion of ΔyjiY because of lack of flagella. The in vivo significance of yjiY was established when ΔyjiY showed decreased colonization of mouse gut. Transcriptome analysis showed upregulation of the virulence factor mgtC in ΔyjiY, which led to better proliferation of ΔyjiY inside macrophages. The expression of mgtC is induced in the absence of proline suggesting that yjiY might be involved in transporting proline rich peptides. Therefore, both cst genes are required for the virulence of Salmonella, but in different host systems. When biofilm forming ability of the wild type and cst mutant strains was tested in vitro, only ΔyjiY strain was unable to form biofilm. Confocal microscopy and assessment of rdar morphotype revealed that ΔyjiY strain lacked extracellular polymeric substance (EPS) production, which is an essential component of biofilm matrix. The mechanism behind was found to be the downregulation in the expression of the biofilm master regulator gene, csgD that controls EPS biosynthesis in Salmonella. Conclusively, yjiY is required for EPS biosynthesis and hence biofilm formation in Salmonella.

碩士
國立交通大學
生化工程研究所
93
Post-transcriptional regulation is an important mechanism for controlling gene expression. RNase E, encoded by the rne gene, is a key enzyme that decides the bulk messenger RNA stability in Escherichia coli. Besides, rRNA processing, polycistronic RNA selective expression and DNA replication are also governed by RNase E. Previous studies suggest the RNase E cleavage depends on environmental conditions, such as temperature, growth rate, and medium composition. They affect its affinity to specific substrates due to the change of RNA secondary structure or others assistant factors. Therefore, it is important to understand the effect of condition changes and RNase E expression. In this study, we examined the effects of carbon sources, growth rate and starvation on rne gene expression. The results reveal that carbon source and growth rate participate in modulating rne transcripts decay rate. The rne mRNA was more stable in minimal medium with glucose than with acetate, and the decay rate increased with growth rate. All of these suggested RNase E maintained its optimal cellular concentration. Under starvation conditions, we observed that rne transcripts dramatically degraded and rne promoters activity were inhibited. Moreover, nutrient deprivation down- regulated RNase E concentration by global regulator (p)ppGpp. Indirect evidence suggests that the elongation of mRNA halt-life resulted from starvation adjust the cell in response to the environment changes.