Construction and characterization of insertion/deletion mutations of the tutF,tutD, and tutG genes of Thauera aromatica strain T1

Thauera aromatica T1 was isolated for its ability to use toluene as a sole carbon source under denitrifying conditions. A genetic approach was used to examine the roles of the tutF, tutD, and tutG gene products (part of a single operon) in the metabolism of toluene. The genes were individually deleted from the chromosome and each resulting mutant strain was unable to metabolize toluene. Plasmids carrying individual in-frame gene deletions failed to complement the corresponding chromosomal deletions but did complement chromosomal deletions downstream of the in-frame deletion. Hence, the tutF, tutD, and tutG genes are each essential for toluene metabolism in T. aromatica T1.     

一种适用于链霉菌异源合成基因簇同框缺失的高效方法

【背景】对抗生素生物合成途径的阐明有助于提高目标化合物的产量并开发具有更高活性的新化合物。基因的同框缺失是天然产物生物合成研究的常规手段,通过分析突变菌株积累的中间产物,可以帮助推导天然产物的合成途径及相关基因的功能。天然产物生物合成基因簇的大小一般在20 kb以上,对每个基因进行同框缺失耗时耗力,因此,优化链霉菌来源的基因同框缺失的方法有重要的意义。【目的】基于PCR-targeting重新设计了一套在链霉菌柯斯文库质粒上进行基因同框缺失的方法,实现链霉菌基因在大肠杆菌中快速、高效的基因同框缺失的技术体系。【方法】使用氨苄青霉素抗性基因bla作为PCR-targeting DNA片段的筛选标记,同时使用体外的Pac I酶切和酶连系统代替体内的Flp/FRT系统来介导同框缺失的构建。【结果】利用这种方法,在6 d内完成了米多霉素生物合成基因簇中14个基因的同框缺失。【结论】此方法与传统的PCR-targeting方法相比,构建同框缺失载体的效率明显提高;Pac I识别序列在链霉菌基因组上的稀有性使得此方法在构建抗生素生物合成基因簇必需基因的同框缺失载体上具有普适性。     

Identification and characterization of Di- and tripeptide transporter DtpT of Listeria monocytogenes EGD-e

Listeria monocytogenes is a gram-positive intracellular pathogen responsible for opportunistic infections in humans and animals. Here we identified and characterized the dtpT gene (lmo0555) of L. monocytogenes EGD-e, encoding the di- and tripeptide transporter, and assessed its role in growth under various environmental conditions as well as in the virulence of L. monocytogenes. Uptake of the dipeptide Pro-[14C]Ala was mediated by the DtpT transporter and was abrogated in a DeltadtpT isogenic deletion mutant. The DtpT transporter was shown to be required for growth when the essential amino acids leucine and valine were supplied as peptides. The protective effect of glycine- and proline-containing peptides during growth in defined medium containing 3% NaCl was noted only in L. monocytogenes EGD-e, not in the DeltadtpT mutant strain, indicating that the DtpT transporter is involved in salt stress protection. Infection studies showed that DtpT contributes to pathogenesis in a mouse infection model but has no role in bacterial growth following infection of J774 macrophages. These studies reveal that DptT may contribute to the virulence of L. monocytogenes.     

The htrA (degP) gene of Listeria monocytogenes 10403S is essential for optimal growth under stress conditions

This report describes a mutant of Listeria monocytogenes strain 10403S (serotype 1/2a) with a defective response to conditions of high osmolarity, an environment that L. monocytogenes encounters in some ready-to-eat foods. A library of L. monocytogenes clones mutagenized with Tn917 was generated and scored for sensitivity to 4% NaCl in order to identify genes responsible for growth or survival in elevated-NaCl environments. One of the L. monocytogenes Tn917 mutants, designated strain OSM1, was selected, and the gene interrupted by the transposon was sequenced. A BLAST search with the putative translated amino acid sequence indicated that the interrupted gene product was a homolog of htrA (degP), a gene coding for a serine protease identified as a stress response protein in several gram-positive and gram-negative bacteria. An htrA deletion strain, strain LDW1, was constructed, and the salt-sensitive phenotype of this strain was complemented by introduction of a plasmid carrying the wild-type htrA gene, demonstrating that htrA is necessary for optimal growth under conditions of osmotic stress. Additionally, strain LDW1 was tested for its response to temperature and H(2)O(2) stresses. The results of these growth assays indicated that strain LDW1 grew at a lower rate than the wild-type strain at 44 degrees C but at a rate similar to that of the wild-type strain when incubated at 4 degrees C. In addition, strain LDW1 was significantly more sensitive to a 52 degrees C heat shock than the wild-type strain. Strain LDW1 was also defective in its response to H(2)O(2) challenge at 37 degrees C, since 100 or 150 micro g of H(2)O(2) was more inhibitory for the growth of strain LDW1 than for that of the parent strain. The stress response phenotype observed for strain LDW1 is similar to that observed for other HtrA(-) organisms, which suggests that L. monocytogenes HtrA may play a role in degrading misfolded proteins that accumulate under stress conditions.     

Listeria monocytogenes tyrosine phosphatases affect wall teichoic acid composition and phage resistance

Tyrosine phosphatase (PTP)-like proteins exist in many bacteria and are segregated into two major groups: low molecular weight and conventional. The latter group also has activity as phosphoinositide phosphatases. These two kinds of PTP are suggested to be involved in many aspects of bacterial physiology including stress response, DNA binding proteins, virulence, and capsule/cell wall production. By annotation, Listeria monocytogenes possesses two potential low molecular weight and two conventional PTPs. Using L.?monocytogenes wild-type (WT) strain 10403S, we have created an in-frame deletion mutant lacking all four PTPs, as well as four additional complemented strains harboring each of the PTPs. No major physiological differences were observed between the WT and the mutant lacking all four PTPs. However, the deletion mutant strain was resistant to Listeria phages A511 and P35 and sensitive to other Listeria phages. This was attributed to reduced attachment to the cell wall. The mutant lacking all PTPs was found to lack N-acetylglucosamine in its wall teichoic acid. Phage sensitivity and attachment was rescued in a complemented strain harboring a low molecular weight PTP (LMRG1707).     

Investigation of the role of ZurR in the physiology and pathogenesis of Listeria monocytogenes

Listeria monocytogenes is a Gram positive pathogen that is ubiquitous in the environment. It is a facultative anaerobic rod that causes listeriosis, a disease with potentially lethal consequences for susceptible individuals. During infection, the pathogen is capable of sequestering metal ions to act as vital biocatalysts in cellular processes. The zinc uptake regulator (ZurR) is predicted to coordinate uptake of zinc from the external environment. An in-frame deletion of the zurR gene resulted in a mutant exhibiting a small colony phenotype and a smaller cell size. The zurR mutant was unaffected under conditions of zinc limitation but demonstrated increased sensitivity to toxic levels of zinc. The mutant also demonstrated a significant (1-log) reduction in virulence potential in the murine model of infection. Using a bioinformatic approach, we identified a number of potentially Zur-regulated genes in the genome of L. monocytogenes. Quantitative RT-PCR demonstrated significant de-repression of zurA, lmo0153, and lmo1671 in the zurR mutant background indicating that these putative transporters are ZurR regulated.     

Exposure to salt and organic acids increases the ability of Listeria monocytogenes to invade Caco-2 cells but decreases its ability to survive gastric stress

The effects of environmental stress exposure on Listeria monocytogenes growth and virulence-associated characteristics were investigated. Specifically, we measured the effects of temperature (7 or 37 degrees C), pH (5.5 or 7.4), the presence of salt and organic acids (375 mM NaCl, 8.45 mM sodium diacetate [SD], 275 mM sodium lactate [SL], or a combination of NaCl, SD, and SL), and deletion of sigB, which encodes a key stress response regulator, on the ability of L. monocytogenes to grow, invade Caco-2 cells, and survive exposure to synthetic gastric fluid (pH 2.5 or 4.5). Our results indicate that (i) L. monocytogenes log-phase generation times and maximum cell numbers are not dependent on the alternative sigma factor sigmaB in the presence of NaCl and organic acids at concentrations typically found in foods; (ii) growth inhibition of L. monocytogenes through the addition of organic acids is pH dependent; (iii) the ability of L. monocytogenes to invade Caco-2 cells is affected by growth phase, temperature, and the presence of salt and organic acids, with the highest relative invasion capabilities observed for cells grown with SL or NaCl at 37 degrees C and pH 7.4; (iv) growth of L. monocytogenes in the presence of NaCl, SD, or SL reduces its ability to survive exposure to gastric fluid; and (v) exposure of L. monocytogenes to gastric fluid reduces the enhanced invasiveness caused by growth in the presence of NaCl or SL. These findings suggest that virulence-associated characteristics that determine the L. monocytogenes infectious dose are likely to be affected by food-specific properties (e.g., pH or the presence of salt or organic acid).     

The response regulator ResD modulates virulence gene expression in response to carbohydrates in Listeria monocytogenes

Listeria monocytogenes is a versatile bacterial pathogen that is able to accommodate to diverse environmental and host conditions. Presently, we have identified a L. monocytogenes two-component response regulator, ResD that is required for the repression of virulence gene expression known to occur in the presence of easily fermentable carbohydrates not found inside host organisms. Structurally and functionally, ResD resembles the respiration regulator ResD in Bacillus subtilis as deletion of the L. monocytogenes resD reduces respiration and expression of cydA, encoding a subunit of cytochrome bd. The resD mutation also reduces expression of mptA encoding the EIIABman component of a mannose/glucose-specific PTS system, indicating that ResD controls sugar uptake. This notion was supported by the poor growth of resD mutant cells that was alleviated by excess of selected carbohydrates. Despite the growth deficient phenotype of the mutant in vitro the mutation did not affect intracellular multiplication in epithelial or macrophage cell lines. When examining virulence gene expression we observed traditional induction by charcoal in both mutant and wild-type cells whereas the repression observed in wild-type cells by fermentable carbohydrates did not occur in resD mutant cells. Thus, ResD is a central regulator of L. monocytogenes when present in the external environment.     

Simultaneous detection of multiple mutations in epidermal growth factor receptor based on fluorescence quenching of quantum dots

We have developed a simultaneous detection method for two common mutations in the epidermal growth factor receptor gene based on the fluorescence quenching phenomenon caused by aggregation of CdSe quantum dots. For detection of the in-frame deletion in exon 19 and the L858R point mutation in exon 21, water-soluble CdSe quantum dots with two sizes were functionalized using four different types of probe oligonucleotides. Addition of target oligonucleotides with the deletion mutation in exon 19 into the suspensions caused crosslinking-induced aggregation of green-emitting quantum dots, followed by the fluorescence quenching while that with the L858R point mutation resulted in aggregation of yellow-emitting quantum dots. In addition, targets with both deletion and point mutations caused aggregation of both green- and yellow-emitting quantum dots. This method allows a simultaneous detection of mutations in exon 19 and 21 of EGFR gene in a single experiment. We found that minimum mutant concentration that could be detected by this method was as low as 2% for deletion mutation, and 5% for point mutation. PCR products of EGFR gene were also used to confirm that our method could be used to detect mutation in amplified DNA fragments.     

Identification and characterization of an essential gene in Listeria monocytogenes using an inducible gene expression system

The Listeria monocytogenes gene lmo1594 is a homolog of the Bacillus subtilis cell division gene ezrA. EzrA is a negative regulator of FtsZ ring formation, which is required for efficient cell division as it regulates the frequency and position of Z-rings in the cell and prevents aberrant polar cell division. Previously identified as a putative high pressure (HP) resistance mechanism; conferring enhanced barotolerance when heterologously expressed against an Escherichia coli background; the aim of the current study was to investigate whether lmo1594 plays a role in listerial barotolerance. When the creation of a deletion mutant proved unsuccessful, the role of lmo1594 was addressed by creating a conditional knockout mutant which demonstrated that the gene is in fact essential for cell survival and growth in L. monocytogenes. In order to investigate the effect of lmo1594 on barotolerance, the gene was over-expressed. The over-expression of lmo1594 increased survival levels in L. monocytogenes treated at 300 MPa, but survival levels similar to those of the wild-type strain were observed when treated at a higher pressure (≥400 MPa). In conclusion, this study reveals for the first time that lmo1594 is absolutely essential for listerial cell growth and survival, and also plays an important role in listerial barotolerance.