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Application of the shsp gene, encoding a small heat shock protein, as a food-grade selection marker for lactic acid bacteria.

ABSTRACT: Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60 degrees C or pH 3.5 and in the ability to grow at 52 degrees C. A food-grade cloning system for S. thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant shsp plasmids into different S. thermophilus and Lactococcus lactis strains. Using a recombinant plasmid carrying an erythromycin resistance (Em(r)) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in S. thermophilus (when we were selecting for heat resistance) were determined to be 2.4 x 10(4) and 1.0 x 10(4) CFU/0.5 micro g of DNA, with standard deviations of 0.54 x 10(4) and 0.32 x 10(4), for shsp and Em(r) selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 x 10(4) and 3.8 x 10(3) CFU/0.5 micro g of DNA, with standard deviations of 0.63 x 10(4) and 3.48 x 10(3), for shsp and Em(r) selection, respectively. The applicability of shsp as a selection marker was further demonstrated by constructing S. thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S. thermophilus plasmid as a functional trait.

SUBMITTER: El Demerdash HA

PROVIDER: S-EPMC169115 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Application of the shsp gene, encoding a small heat shock protein, as a food-grade selection marker for lactic acid bacteria.

El Demerdash Hassan A M HA Heller Knut J KJ Geis Arnold A

Applied and environmental microbiology 20030801 8

Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60 degrees C or pH 3.5 a ...[more]

PMID: 12902223

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Project description:Whole-genome sequencing has revolutionized and accelerated scientific research that aims to study the genetics, biochemistry and molecular biology of bacteria. Lactic acid-producing bacteria, which include lactic acid bacteria (LAB) and bifidobacteria, are typically Gram-positive, catalase-negative organisms, which occupy a wide range of natural plant- and animal-associated environments. LAB species are frequently involved in the transformation of perishable raw materials into more stable, pleasant, palatable and safe fermented food products. LAB and bifidobacteria are also found among the resident microbiota of the gastrointestinal and/or genitourinary tracts of vertebrates, where they are believed to exert health-promoting effects. At present, the genomes of more than 20 LAB and bifidobacterial species have been completely sequenced. Their genome content reflects its specific metabolism, physiology, biosynthetic capabilities, and adaptability to varying conditions and environments. The typical LAB/bifidobacterial genome is relatively small (from 1.7 to 3.3 Mb) and thus harbors a limited assortment of genes (from around 1,600 to over 3,000). These small genomes code for a broad array of transporters for efficient carbon and nitrogen assimilation from the nutritionally-rich niches they usually inhabit, and specify a rather limited range of biosynthetic and degrading capabilities. The variation in the number of genes suggests that the genome evolution of each of these bacterial groups involved the processes of extensive gene loss from their particular ancestor, diversification of certain common biological activities through gene duplication, and acquisition of key functions via horizontal gene transfer. The availability of genome sequences is expected to revolutionize the exploitation of the metabolic potential of LAB and bifidobacteria, improving their use in bioprocessing and their utilization in biotechnological and health-related applications.

Project description:BackgroundIn living organisms, small heat shock proteins (sHSPs) are triggered in response to stress situations. This family of proteins is large in plants and, in the case of tomato (Solanum lycopersicum), 33 genes have been identified, most of them related to heat stress response and to the ripening process. Transcriptomic and proteomic studies have revealed complex patterns of expression for these genes. In this work, we investigate the coregulation of these genes by performing a computational analysis of their promoter architecture to find regulatory motifs known as heat shock elements (HSEs). We leverage the presence of sHSP members that originated from tandem duplication events and analyze the promoter architecture diversity of the whole sHSP family, focusing on the identification of HSEs.ResultsWe performed a search for conserved genomic sequences in the promoter regions of the sHSPs of tomato, plus several other proteins (mainly HSPs) that are functionally related to heat stress situations or to ripening. Several computational analyses were performed to build multiple sequence motifs and identify transcription factor binding sites (TFBS) homologous to HSF1AE and HSF21 in Arabidopsis. We also investigated the expression and interaction of these proteins under two heat stress situations in whole tomato plants and in protoplast cells, both in the presence and in the absence of heat shock transcription factor A2 (HsfA2). The results of these analyses indicate that different sHSPs are up-regulated depending on the activation or repression of HsfA2, a key regulator of HSPs. Further, the analysis of protein-protein interaction between the sHSP protein family and other heat shock response proteins (Hsp70, Hsp90 and MBF1c) suggests that several sHSPs are mediating alternative stress response through a regulatory subnetwork that is not dependent on HsfA2.ConclusionsOverall, this study identifies two regulatory motifs (HSF1AE and HSF21) associated with the sHSP family in tomato which are considered genomic HSEs. The study also suggests that, despite the apparent redundancy of these proteins, which has been linked to gene duplication, tomato sHSPs showed different up-regulation and different interaction patterns when analyzed under different stress situations.

Project description:Backcrossing together with simple sequence repeat marker strategy was adopted to improve popular Malaysian chilli Kulai (Capsicum annuum L.) for heat tolerance. The use of molecular markers in backcross breeding and selection contributes significantly to overcoming the main drawbacks such as increase linkage drag and time consumption, in the ancient manual breeding approach (conventional), and speeds up the genome recovery of the recurrent parent. The strategy was adopted to introgress heat shock protein gene(s) from AVPP0702 (C. annuum L.), which are heat-tolerant, into the genetic profile of Kulai, a popular high-yielding chilli but which is heat sensitive. The parents were grown on seed trays, and parental screening was carried out with 252 simple sequence repeat markers. The selected parents were crossed and backcrossed to generate F1 hybrids and backcross generations. Sixty-eight markers appeared to be polymorphic and were used to assess the backcross generation; BC1F1, BC2F1 and BC3F1. The average recipient allele of the selected four BC1F1 plants was 80.75% which were used to produce the BC2F1 generation. BC1-P7 was the best BC1F1 plant because it had the highest recovery at 83.40% and was positive to Hsp-linked markers (Hsp70-u2 and AGi42). After three successive generations of backcrossing, the average genome recovery of the recurrent parent in the selected plants in BC3F1 was 95.37%. Hsp gene expression analysis was carried out on BC1F1, BC2F1 and BC3F1 selected lines. The Hsp genes were found to be up-regulated when exposed to heat treatment. The pattern of Hsp expression in the backcross generations was similar to that of the donor parent. This confirms the successful introgression of a stress-responsive gene (Hsp) into a Kulai chilli pepper variety. Furthermore, the yield performance viz. plant height, number of fruits, fruit length and weight and total yield of the improved plant were similar with the recurrent parent except that the plant height was significantly lower than the Kulai (recurrent) parent.

Dataset Information

Application of the shsp gene, encoding a small heat shock protein, as a food-grade selection marker for lactic acid bacteria.

Publications

Application of the shsp gene, encoding a small heat shock protein, as a food-grade selection marker for lactic acid bacteria.

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