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Differential expression of the HvCslF6 gene late in grain development may explain quantitative differences in (1,3;1,4)-β-glucan concentration in barley

Wong SC, Shirley NJ, Little A, Khoo KH, Schwerdt J, Fincher GB, et al.

Molecular breeding : new strategies in plant improvement, 2015 01 20;35:20.
PMID: 25620877

The cellulose synthase-like gene HvCslF6, which is essential for (1,3;1,4)-β-glucan biosynthesis in barley, collocates with quantitative trait loci (QTL) for grain (1,3;1,4)-β-glucan concentration in several populations, including CDC Bold × TR251. Here, an alanine-to-threonine substitution (caused by the only non-synonymous difference between the CDC Bold and TR251 HvCslF6 alleles) was mapped to a position within HvCSLF6 that seems unlikely to affect enzyme stability or function. Consistent with this, transient expression of full-length HvCslF6 cDNAs from CDC Bold and TR251 in Nicotianabenthamiana led to accumulation of similar amounts of (1,3;1,4)-β-glucan accumulation. Monitoring of HvCslF6 transcripts throughout grain development revealed a significant difference late in grain development (more than 30 days after pollination), with TR251 [the parent with higher grain (1,3;1,4)-β-glucan] exhibiting higher transcript levels than CDC Bold. A similar difference was observed between Beka and Logan, the parents of another population in which a QTL had been mapped in the HvCslF6 region. Sequencing of a putative promoter region of HvCslF6 revealed numerous polymorphisms between CDC Bold and TR251, but none between Beka and Logan. While the results of this work indicate that naturally occurring quantitative differences in (1,3;1,4)-β-glucan accumulation may be due to cis-regulated differences in HvCslF6 expression, these could not be attributed to any specific DNA sequence polymorphism. Nevertheless, information on HvCslF6 sequence polymorphism was used to develop molecular markers that could be used in barley breeding to select for the desired [low or high (1,3;1,4)-β-glucan] allele of the QTL.

Matched MeSH terms: Glucosyltransferases
Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function

Goh KM, Liew KJ, Chai KP, Illias RM

Methods Mol Biol, 2017;1498:385-396.
PMID: 27709591

Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.

Matched MeSH terms: Glucosyltransferases/genetics*
Comparative studies on the effect of crude aqueous (CA) and solvent (CM) extracts of clove on the cariogenic properties of Streptococcus mutans

Rahim ZH, Khan HB

J Oral Sci, 2006 Sep;48(3):117-23.
PMID: 17023743

A study was conducted to compare the efficiency of crude aqueous (CA) and solvent extracts (CM) of clove on the caries-inducing properties of Streptococcus mutans. The cariogenic properties investigated included the cell adhesion, cell-surface hydrophobicity and glucan synthesis activities of S. mutans. There was a significant difference between the effect of the CA and CM extracts on the adhesion of S. mutans (P < 0.05) within a concentration range of 5-15 mg/ml, the CM extract demonstrating a slightly higher inhibitory effect. However, the effect of the CM extract on the cell-surface hydrophobicity of S. mutans was weaker than that of the CA extract. The two extracts were found to reduce the synthesis of water-insoluble glucan (WIG) by almost 50% at a concentration as low as 0.5 mg/ml and the CM extract exhibited a significantly higher inhibitory effect than the CA extract (P < 0.05). The present findings indicate that both the CA and CM extracts exert inhibitory effects on the cariogenic properties of S. mutans and that the CA extract is as equally effective as the CM extract.

Matched MeSH terms: Glucosyltransferases/antagonists & inhibitors
Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system

Low KO, Mahadi NM, Abdul Rahim R, Rabu A, Abu Bakar FD, Abdul Murad AM, et al.

J Biotechnol, 2010 Dec;150(4):453-9.
PMID: 20959127 DOI: 10.1016/j.jbiotec.2010.10.001

The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.

Matched MeSH terms: Glucosyltransferases/secretion*
Fulltext Shigella flexneri serotype 1c derived from serotype 1a by acquisition of gtrIC gene cluster via a bacteriophage

Tang SS, Carlin NI, Talukder KA, Cam PD, Verma NK

BMC Microbiol, 2016 Jun 27;16(1):127.
PMID: 27349637 DOI: 10.1186/s12866-016-0746-z

BACKGROUND: Shigella spp. are the primary causative agents of bacillary dysentery. Since its emergence in the late 1980s, the S. flexneri serotype 1c remains poorly understood, particularly with regard to its origin and genetic evolution. This article provides a molecular insight into this novel serotype and the gtrIC gene cluster that determines its unique immune recognition.
RESULTS: A PCR of the gtrIC cluster showed that serotype 1c isolates from different geographical origins were genetically conserved. An analysis of sequences flanking the gtrIC cluster revealed remnants of a prophage genome, in particular integrase and tRNA(Pro) genes. Meanwhile, Southern blot analyses on serotype 1c, 1a and 1b strains indicated that all the tested serotype 1c strains may have had a common origin that has since remained distinct from the closely related 1a and 1b serotypes. The identification of prophage genes upstream of the gtrIC cluster is consistent with the notion of bacteriophage-mediated integration of the gtrIC cluster into a pre-existing serotype.
CONCLUSIONS: This is the first study to show that serotype 1c isolates from different geographical origins share an identical pattern of genetic arrangement, suggesting that serotype 1c strains may have originated from a single parental strain. Analysis of the sequence around the gtrIC cluster revealed a new site for the integration of the serotype converting phages of S. flexneri. Understanding the origin of new pathogenic serotypes and the molecular basis of serotype conversion in S. flexneri would provide information for developing cross-reactive Shigella vaccines.

Matched MeSH terms: Glucosyltransferases/genetics
Isolation and characterization of full-length cellulose synthase gene (HsCesA1) from roselle (hibiscus sabdariffa l. Var. UMKL)

Seyedi SS, Tan SG, Namasivayam P, Yong CSY

Sains Malaysiana, 2016;45:717-727.

The Hibiscus sabdariffa var. UMKL (Roselle) investigated here may potentially be used as an alternative fibre source. To
the best of our knowledge, there was no study focusing on the genetics underlying the cellulose biosynthesis machinery
in Roselle thus far. This paper presents the results of the first isolation of the cellulose synthase gene, HsCesA1 from this
plant, which is fundamental for working towards understanding the functions of CesA genes in the cellulose biosynthesis
of Roselle. A full-length HsCesA1 cDNA of 3528 bp in length (accession no: KJ608192) encoding a polypeptide of 974
amino acid was isolated. The full-length HsCesA1 gene of 5489 bp length (accession no: KJ661223) with 11-introns
and a promoter region of 737 bp was further isolated. Important and conserved characteristics of a CesA protein were
identified in the HsCesA1 deduced amino acid sequence, which strengthened the prediction that the isolated gene being
a cellulose synthase belonging to the processive class of the 2-glycosyltransferase family 2A. Relative gene expression
analysis by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) on young leaf and stem tissues
found that HsCesA1 had similar levels of gene expression in both tissues. Phylogenetic and Blast analyses also supported
the prediction that the isolated HsCesA1 may play roles in the cell wall depositions in both leaf and stem tissues.

Matched MeSH terms: Glucosyltransferases