Soon-Kwang Hong

Agar degradation by microorganisms and agar-degrading enzymes

Agar is a mixture of heterogeneous galactans, mainly composed of 3,6-anhydro-l-galactoses (or l-galactose-6-sulfates) d-galactoses and l-galactoses (routinely in the forms of 3,6-anhydro-l-galactoses or l-galactose-6-sulfates) alternately linked by β-(1,4) and α-(1,3) linkages. It is a major component of the cell walls of red algae and has been used in a variety of laboratory and industrial applications, owing to its jellifying properties. Many microorganisms that can hydrolyze and metabolize agar as a carbon and energy source have been identified in seawater and marine sediments. Agarolytic microorganisms commonly produce agarases, which catalyze the hydrolysis of agar. Numerous agarases have been identified in microorganisms of various genera. They are classified according to their cleavage pattern into three types—α-agarase, β-agarase, and β-porphyranase. Although, in a broad sense, many other agarases are involved in complete hydrolysis of agar, most of those identified are β-agarases. In this article we review agarolytic microorganisms and their agar-hydrolyzing systems, covering β-agarases as well as α-agarases, α-neoagarobiose hydrolases, and β-porphyranases, with emphasis on the recent discoveries. We also present an overview of the biochemical and structural characteristics of the various types of agarases. Further, we summarize and compare the agar-hydrolyzing systems of two specific microorganisms: Gram-negative Saccharophagus degradans 2–40 and Gram-positive Streptomyces coelicolor A3(2). We conclude with a brief discussion of the importance of agarases and their possible future application in producing oligosaccharides with various nutraceutical activities and in sustainably generating stock chemicals for biorefinement and bioenergy.

Primary structure of AfsR, a global regulatory protein for secondary metabolite formation in Streptomyces coelicolor A3(2)

The afsR gene of Streptomyces coelicolor A3(2) complements afsB mutations affecting production of pigmented antibiotics. It also directs pigment production in Streptomyces lividans when carried on a plasmid vector. Nucleotide sequencing of the afsR gene revealed that it codes for a 993-amino acid protein (Mr 105,600) with A- and B-type ATP-binding consensus sequences at its N-terminal portion and two DNA-binding consensus sequences with a helix-turn-helix motif at its C-terminal portion. Each of the N- and C-terminal halves was capable of conferring pigment production, to some extent, in S. lividans, when carried separately on a multicopy plasmid. In addition, expression in trans of the two regions on the same plasmid conferred pigment production to almost the same extent as did the intact afsR gene. Mutations at the two ATP-binding consensus sequences, that were generated by in vitro site-directed mutagenesis, revealed their functional importance. Disruption of the S. coelicolor A3(2) chromosomal afsR gene in either the N- or C-terminal half using phage phi C31 KC515 resulted in significant, but not complete, loss of pigment production. These data suggest that the AfsR protein comprises two domains, viz., an ATP-binding and a DNA-binding domain, each of which could function as a positive regulator for pigment production. These afsR mutants sporulate normally. In addition to an internal promoter, which we previously detected in the middle of the AfsR coding region, S1 nuclease mapping revealed two tandem transcriptional start points, separated by 64 bp, upstream from a putative ATG start codon of the AfsR product.

Accumulation of S-Adenosyl-l-Methionine Enhances Production of Actinorhodin but Inhibits Sporulation in Streptomyces lividans TK23

S-Adenosyl-L-methionine synthetase (SAM-s) catalyzes the biosynthesis of SAM from ATP and L-methionine. Despite extensive research with many organisms, its role in Streptomyces sp. remains unclear. In the present study, the putative SAM-s gene was isolated from a spectinomycin producer, Streptomyces spectabilis. The purified protein from the transformed Escherichia coli with the isolated gene synthesized SAM from L-methionine and ATP in vitro, strongly indicating that the isolated gene indeed encoded the SAM-s protein. The overexpression of the SAM-s gene in Streptomyces lividans TK23 inhibited sporulation and aerial mycelium formation but enhanced the production of actinorhodin in both agar plates and liquid media. Surprisingly, the overexpressed SAM was proven by Northern analysis to increase the production of actinorhodin through the induction of actII-ORF4, a transcription activator of actinorhodin biosynthetic gene clusters. In addition, we found that a certain level of intracellular SAM is critical for the induction of antibiotic biosynthetic genes, since the control strain harboring only the plasmid DNA did not show any induction of actII-ORF4 until it reached a certain level of SAM in the cell. From these results, we concluded that the SAM plays important roles as an intracellular factor in both cellular differentiation and antibiotic production in Streptomyces sp.

Identification of an A-factor-dependent promoter in the streptomycin biosynthetic gene cluster of Streptomyces griseus

SummaryA-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is a microbial hormone controlling streptomycin (Sm) production, Sm resistance and sporulation in Streptomyces griseus. In order to identify A-factor-dependent promoters in the Sm biosynthetic gene cluster, a new promoter-probe plasmid with a low copy number was constructed by using an extremely thermostable malate dehydrogenase gene as the reporter. Of the three promoters in the Sm production region that includes strR, aphD and strB, only the promoter of strR, which codes for a putative regulatory protein, was found to be directly controlled by A-factor. This was also confirmed by S1 nuclease mapping. The region essential for its A-factor-dependence was determined to be located 430–330 base pairs upstream of the transcriptional start point. Increase in the copy number of the strR promoter region did not lead to a corresponding increase in the total promoter activity, probably due to titration of a putative activator which binds to the enhancer-like region and controls the expression of the strR promoter. This putative activator is apparently distinct from the A-factor-receptor protein. The aphD gene, which encodes the major Sm resistance determinant, Sm-6-phosphotransferase, was transcribed mainly by read-through from the A-factor-dependent strR promoter; this accounts for the prompt induction of Sm resistance by A-factor.

Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus

SummaryIn vitro phosphorylation reactions using extracts of Streptomyces griseus cells and γ-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.

Transcriptional control by A-factor of two trypsin genes in Streptomyces griseus.

AdpA is the key transcriptional activator for a number of genes of various functions in the A-factor regulatory cascade in Streptomyces griseus, forming an AdpA regulon. Trypsin-like activity was detected at a late stage of growth in the wild-type strain but not in an A-factor-deficient mutant. Consistent with these observations, two trypsin genes, sprT and sprU, in S. griseus were found to be members of the AdpA regulon; AdpA activated the transcription of both genes by binding to the operators located at about -50 nucleotide positions with respect to the transcriptional start point. The transcription of sprT and sprU, induced by AdpA, was most active at the onset of sporulation. Most trypsin activity exerted by S. griseus was attributed to SprT, because trypsin activity in an sprT-disrupted mutant was greatly reduced but that in an sprU-disrupted mutant was only slightly reduced. This was consistent with the observation that the amount of the sprT mRNA was much greater than that of the sprU transcript. Disruption of both sprT and sprU (mutant DeltasprTU) reduced trypsin activity to almost zero, indicating that no trypsin genes other than these two were present in S. griseus. Even the double mutant DeltasprTU grew normally and developed aerial hyphae and spores over the same time course as the wild-type strain.

Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2): an exo- and endo-type β-agarase-producing neoagarobiose

Streptomyces coelicolor can degrade agar, the main cell wall component of red macroalgae, for growth. To constitute a crucial carbon source for bacterial growth, the alternating α-(1,3) and β-(1,4) linkages between the 3,6-anhydro-L-galactoses and D-galactoses of agar must be hydrolyzed by α/β-agarases. In S. coelicolor, DagA was confirmed to be an endo-type β-agarase that degrades agar into neoagarotetraose and neoagarohexaose. Genomic sequencing data of S. coelicolor revealed that Sco3487, annotated as a putative hydrolase, has high similarity to the glycoside hydrolase (GH) GH50 β-agarases. Sco3487 encodes a primary translation product (88.5 kDa) of 798 amino acids, including a 45-amino-acid signal peptide. The sco3487 gene was cloned and expressed under the control of the ermE promoter in Streptomyces lividans TK24. β-Agarase activity was detected in transformant culture broth using the artificial chromogenic substrate p-nitrophenyl-β-D-galactopyranoside. Mature Sco3487 (83.9 kDa) was purified 52-fold with a yield of 66% from the culture broth. The optimum pH and temperature for Sco3487 activity were 7.0 and 40°C, respectively. The K(m) and V(max) for agarose were 4.87 mg/ml (4 × 10(-5) M) and 10.75 U/mg, respectively. Sco3487 did not require metal ions for its activity, but severe inhibition by Mn(2+) and Cu(2+) was observed. Thin-layer chromatography analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and Fourier transform-nuclear magnetic resonance spectrometry of the Sco3487 hydrolysis products revealed that Sco3487 is both an exo- and endo-type β-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose.

pH shock induces overexpression of regulatory and biosynthetic genes for actinorhodin productionin Streptomyces coelicolor A3(2)

Actinorhodin production is markedly enhanced when an acidic pH shock is applied to a surface-grown culture of Streptomyces coelicolor A3(2). For an in-depth study of this phenomenon, transcriptional analyses using DNA microarrays and reverse transcription polymerase chain reaction and proteomic analysis were performed. Investigated were expression levels of the regulators and enzymes responsible for signal transduction and actinorhodin biosynthesis and enzymes involved in some major metabolic pathways. Regulators PkaG, AfsR, AfsS and/or another unidentified regulator and ActII-ORF4, in sequence, were observed to be activated by pH shock. In addition, a number of genes associated with actinorhodin production and secretion and the major central metabolic pathways investigated were observed to be upregulated with pH shock. Fatty acid degradation was particularly promoted by pH shock, while fatty acid biosynthesis was suppressed; it is envisaged that this enriches the precursor pool (acetyl-CoA) and building blocks for actinorhodin biosynthesis. Furthermore, glucose 6-phosphate dehydrogenases, initiating the pentose phosphate pathway, were highly activated by pH shock, enriching the reduced nicotinamide adenine dinucleotide phosphate (NADPH) pool for biosynthesis in general. It is deduced that these metabolic changes caused by pH shock have positively contributed to the stimulation of actinorhodin biosynthesis in a concerted manner.

Acidic pH shock induces the expressions of a wide range of stress-response genes

BackgroundEnvironmental signals usually enhance secondary metabolite production in Streptomycetes by initiating complex signal transduction system. It is known that different sigma factors respond to different types of stresses, respectively in Streptomyces strains, which have a number of unique signal transduction mechanisms depending on the types of environmental shock. In this study, we wanted to know how a pH shock would affect the expression of various sigma factors and shock-related proteins in S. coelicolor A3(2).ResultsAccording to the results of transcriptional and proteomic analyses, the major number of sigma factor genes were upregulated by an acidic pH shock. Well-studied sigma factor genes of sigH (heat shock), sigR (oxidative stress), sigB (osmotic shock), and hrdD that play a major role in the secondary metabolism, were all strongly upregulated by the pH shock. A number of heat shock proteins including the DnaK family and chaperones such as GroEL2 were also observed to be upregulated by the pH shock, while their repressor of hspR was strongly downregulated. Oxidative stress-related proteins such as thioredoxin, catalase, superoxide dismutase, peroxidase, and osmotic shock-related protein such as vesicle synthases were also upregulated in overall.ConclusionFrom these observations, an acidic pH shock was considered to be one of the strongest stresses to influence a wide range of sigma factors and shock-related proteins including general stress response proteins. The upregulation of the sigma factors and shock proteins already found to be related to actinorhodin biosynthesis was considered to have contributed to enhanced actinorhodin productivity by mediating the pH shock signal to regulators or biosynthesis genes for actinorhodin production.

Identification and Characterization of a Xyloglucan-Specific Family 74 Glycosyl Hydrolase from Streptomyces coelicolor A3(2)

ABSTRACT The sco6545 gene of Streptomyces coelicolor A3(2) was nominated as a putative cellulase with 863 mature-form amino acids (90.58 kDa). We overexpressed and purified Sco6545 and demonstrated that the protein is not a cellulase but a xyloglucan-specific glycosyl hydrolase which cleaves xyloglucan at unbranched glucose residues.