γ-Butyrolactones in
Streptomyces
are well recognized as bacterial hormones, and they affect secondary metabolism of
Streptomyces
. γ-Butyrolactone receptors are considered important regulatory ...proteins, and various γ-butyrolactone synthases and receptors have been reported in
Streptomyces
. Here, we characterized a new regulator, SCO0608, that interacted with SCB1 (γ-butyrolactone of
Streptomyces coelicolor
) and bound to the
scbR/A
and
adpA
promoters. The SCO0608 protein sequences are not similar to those of any known γ-butyrolactone binding proteins in
Streptomyces
such as ScbR from
S. coelicolor
or ArpA from
Streptomyces griseus
. Interestingly, SCO0608 functions as a repressor of antibiotic biosynthesis and spore formation in R5 complex media. We showed the existence of another type of γ-butyrolactone receptor in
Streptomyces
, and this SCO0608 was named ScbR-like γ-butyrolactone binding regulator (SlbR) in
S. coelicolor
.
Amylases are important industrial enzymes that have been applied widely in the food, detergent, and pulp industries and fermentation processes. In the present study, a gene encoding an alpha-amylase ...from the genomic DNA library of
Paenibacillus
sp. was identified and characterized. The amylase gene designated
amy1
was shown to consist of 1,980 bp and shared sequence identity towards α-amylase genes from other
Bacillus
sp. The deduced amino acid sequence for Amy1 indicated 80 % sequence identity with other
Bacillus
strains. Heterologous expression of recombinant Amy1 in
Escherichia coli
BL21(DE3) facilitated the recovery of this protein in soluble form. Enzyme kinetic data revealed Amy1 to have a
K
m
of 23.83 mg/mL and
K
cat
of 48.74 min
−1
and
K
cat
/K
m
of 2 min
−1
mg
−1
mL
−1
for starch. The activity of this protein was found to be enhanced by Mn
2+
, and furthermore, Amy1 remained active at a broad pH range (4–10) and temperature (30–90 °C). The ability of Amy1 to act on food waste under broad temperature and pH conditions, together with its ability to produce simple sugars, shows many advantages for further application in the food industry.
Inhibitors of 3OC12, an initial signal molecule of the quorum sensing (QS) signaling cascade in Pseudomonas aeruginosa have been developed. Eight inhibitor candidates were synthesized by substituting ...the head part of 3-oxododecanoyl-homoserine lactone (3OC12) with different aromatic rings, and their docking poses and scores (binding energies) were predicted by in silico modeling study. All compounds gave better docking scores than 3OC12 and good inhibition effects on LasR activity in the in vivo bioassay. Like the modifications in the tail part of 3OC12 in our previous study Kim et al. (2008), the head-part modifications also showed inhibition activity in a fairly good proportion to the docking scores from the modeling analysis. This implies that the head part of 3OC12 also contributes significantly to forming the active conformation of the LasR-3OC12 complex, and its modification could effectively induce the inactive conformation of the complex. We suggest that the head part of 3OC12 is also a good target moiety to develop the structure-based Pseudomonas QS inhibitors.
Several reports state that three architectural units, including integration host factor, leucyl aminopeptidase (PepA), and purine regulator, are involved in transcriptional process with RNA ...polymerase in
Escherichia coli
. Similarly,
Streptomyces
species possess the same structural units. We previously identified a protein,
Streptomyces
integration host factor (sIHF), involved in antibiotic production and sporulation. Subsequently, the function of PepA (SCO2179) was examined in detail. PepA is highly conserved among various
Streptomyces
spp., but it has not yet been characterized in
Streptomyces coelicolor
. While it is annotated as a putative leucyl aminopeptidase because it contains a peptidase M17 superfamily domain, this protein did not exhibit leucyl aminopeptidase activity. SCO2179 deletion mutant showed increased actinorhodin production and sporulation, as well as more distinct physiological differences, particularly when cultured on
N
-acetylglucosamine (GlcNAc) minimal media. The results of two-dimensional gel analysis and reverse transcription PCR showed that the SCO2179 deletion increased protein and mRNA levels of
ftsZ
,
ssgA
, and actinorhodin (ACT)-related genes such as
actII
-
ORF4
, resulting in increased actinorhodin production and spore formation in minimal media containing GlcNAc.
Background: Painful plantar callosities under lesser metatarsal heads are commonly associated with hallux valgus. The purpose of the present study was to evaluate the prognosis of painful plantar ...callosities after hallux valgus correction without lesser metatarsal osteotomy in hallux valgus deformity. Materials and Methods: Between September 2004 and June 2007, 31 patients (40 feet) underwent proximal chevron first metatarsal osteotomy with a distal soft tissue procedure, with preoperatively painful plantar callosities under lesser metatarsal heads. Clinical results were evaluated using a visual analogue scale (VAS), the American Orthopaedic Foot and Ankle Society (AOFAS) hallux-metatarsophalangeal interphalangeal scales, and a modified 70-point clinical scale. Radiographic evaluations included hallux valgus angle and intermetatarsal angle. Results: Thirty-two (80%) of the 40 feet had no pain and callosity and 5 (12.5%) had no pain but residual plantar callosities, and 3 (7.5%) were not improved at final evaluation. The mean VAS and AOFAS scores were improved from 7.8 ± 1.6 to 1.9 ± 1.5 points and from 53.8 ± 14.2 to 92.6 ± 15.3 points, respectively. In terms of the 70-point clinical scale, overall clinical results were good in 34 feet and fair in 6. The mean hallux valgus and intermetatarsal angles were improved from 36.6 ± 6.2 to 12.5 ± 5.9 degrees and from 17.5 ± 3.9 to 8.6 ± 3.5 degrees, respectively. Conclusion: Painful plantar callosities under the lesser metatarsals in patients with hallux valgus deformity can be improved by hallux valgus correction alone without lesser metatarsal osteotomy.
Level of Evidence: IV, Retrospective Case Series
Pick and choose: The identification of the substrate specificity of a protein kinase is critical in understanding its role and function in a cellular signal transduction network. A high‐throughput ...platform was developed for the identification of tyrosine kinase substrate specificity by using an improved one‐bead‐one‐compound ladder peptide library and MALDI‐TOF MS.
Modification of chloramphenicol to a phosphorylated derivative mediated by chloramphenicol phosphotransferase (Yhr2) from Streptomyces avermitilis MA4680.
Although phosphorylation of chloramphenicol ...has been shown to occur in the chloramphenicol producer, Streptomyces venezuelae, there are no reports on the existence of chloramphenicol phosphorylase in other Streptomyces species. In the present study, we report the modification of chloramphenicol by a recombinant protein, designated as Yhr2 (encoded by SAV_877), from Streptomyces avermitilis MA4680. Recombinant Yhr2 was expressed in Escherichia coli BL21 (DE3) and the cells expressing this recombinant protein were shown to phosphorylate chloramphenicol to a 3′-O-phosphoryl ester derivative, resulting in an inactivated form of the antibiotic. Expression of yhr2 conferred chloramphenicol resistance to E. coli cells up to 25μg/mL and in an in vitro reaction, adenosine triphosphate (ATP), guanosine triphosphate (GTP), adenosine diphosphate (ADP) and guanosine diphosphate (GDP) were shown to be the phosphate donors for phosphorylation of chloramphenicol. This study highlights that antibiotic resistance conferring genes could be easily expressed and functionalized in other organisms that do not produce the respective antibiotic.
Phosphomannose isomerases (PMIs) in bacteria and fungi catalyze the reversible conversion of
d
-fructose-6-phosphate to
d
-mannose-6-phosphate during biosynthesis of GDP-mannose, which is the main ...intermediate in the mannosylation of important cell wall components, glycoproteins, and certain glycolipids. In the present study, the kinetic parameters of PMI from
Streptomyces coelicolor
were obtained, and its function on antibiotic production and sporulation was studied.
manA
(SCO3025) encoding PMI in
S
.
coelicolor
was deleted by insertional inactivation. Its mutant (
S
.
coelicolor
∆
manA
) was found to exhibit a
bld
-like phenotype. Additionally,
S
.
coelicolor
∆
manA
failed to produce the antibiotics actinorhodin and red tripyrolle undecylprodigiosin in liquid media. To identify the function of
manA
, the gene was cloned and expressed in
Escherichia coli
BL21 (DE3). The purified recombinant ManA exhibited PMI activity (
K
cat
/
K
m
(mM
−1
s
−1
= 0.41 for
d
-mannose-6-phosphate), but failed to show GDP-
d
-mannose pyrophosphorylase GMP (ManC) activity. Complementation analysis with
manA
from
S
.
coelicolor
or
E
.
coli
resulted in the recovery of
bld
-like phenotype of
S
.
coelicolor
∆
manA
. SCO3026, another ORF that encodes a protein with sequence similarity towards bifunctional PMI and GMP, was also tested for its ability to function as an alternate ManA. However, the purified protein of SCO3026 failed to exhibit both PMI and GMP activity. The present study shows that enzymes involved in carbohydrate metabolism could control cellular differentiation as well as the production of secondary metabolites.
SCO0948 was found to be the single open reading frame annotated to encode an α-mannosidase (AM1) in Streptomyces coelicolor M145. To characterize the protein, we overexpressed SCO0948 in Escherichia ...coli BL21(DE3). Recombinant AM1, with a molecular weight of 110 kDa, exhibited α-mannosidase activity toward 4-nitrophenyl-α-D-mannopyranoside with a K ₘ of 4.61 mM, a V ₘₐₓ of 101.6 mM/min, and a specific activity of 47.96 U/mg. Treatment of ovalbumin, a glycoprotein, with AM1 resulted in partial deglycosylation, as assessed by glycostaining and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The S. coelicolor deletion mutant for SCO0948 failed to produce α-mannosidase activity, confirming AM1 as the only α-mannosidase in S. coelicolor M145. Interestingly, the deletion mutant and a complementation strain produced lower levels of the antibiotics actinorhodin and undecylprodigiosin in glucose minimal media. The results indicate that AM1 as an α-mannosidase influences deglycosylation and antibiotic production in S. coelicolor M145.
In the process of evaluating the growth of Streptomyces coelicolor on rich media such as blood agar, we found that S. coelicolor a non-pathogenic, well-known antibiotic producer had the ability to ...grow and produce a prominent hemolytic zone. By comparing the growth with an agarase gene mutant of S. coelicolor, a similar prominent hemolytic zone was found to develop due to the organism's hemolytic activity. After the confirmation of hemolytic activity from S. coelicolor, the genome was searched for hemolysin-coding genes; consequently, SCO1782, SCO2534, and SCO3882 were identified, whose products were annotated as a putative, membrane, and hypothetical proteins, respectively. Functional characterization of all the recombinant proteins expressed in Escherichia coli BL21(DE3) revealed that only SCO1782 exhibited hemolytic activity. This S. coelicolor protein, designated as S-hemolysin, showed sequence similarity toward hemolysins from Brachyspira hyodysenteriae (35%) and Mycobacterium tuberculosis (62%). Recombinant hemolysin exhibited activity against sheep blood erythrocytes and cytolytic activity against human fibroblast cells. Deletion of SCO1782 resulted in complete loss of hemolysin activity in S. coelicolor.
•The first report on a hemolysin from a well studied antibiotics producer, Streptomyces coelicolor.•Functional expression and purification of S-Hemolysin from S. coelicolor in E. coli.•S-Hemolysin exhibiting hemolytic and cellulolytic activity against Eukaryotic cells.
