In previous paper we reported that Streptomyces sp. strain MBR-52 had a potential to accelerate rooting of cucumber seedlings and tissue-cultured seedlings of rhododendron. The carrier of this strain for soil application is one of the key factors for successful agricultural use. For such a practical purpose, organic carriers such as cereal bran, sawdust, and a variety of polysaccharides are routinely used. However, they often cause undesirable adverse effects on plant growth. Thus, the ceramic bead was preferentially selected as a carrier candidate in this paper. MBR-52 successfully attached the beads when they were immersed in mycelial suspension of this strain in culture medium, followed by air-drying. Microscopy demonstrated that mycelia of MBR-52 expanded on the bead surface forming numerous spores. This strain continued to survive on the beads at least 5 weeks when the beads were stocked at −20 to 25°C if the prior incubation at 25°C allowed the strain to colonize well and sporulate before the stock. When tissue-cultured seedlings of mountain laurel were transplanted into soil where the beads with colonizing MBR-52 were buried, rooting from the seedlings was apparently accelerated. MBR-52 was able to be re-isolated from these seedlings, confirming its endophytic colonization. X-ray microanalysis revealed that minerals required for growth of MBR-52 were detected in the bead. Thus, MBR-52 could survive using medium remnants covering the beads and essential inorganic minerals supplied from the beads.
In the present study, we tried to isolate bacterial strains from inside soil aggregates as generally microbial flora inside differs from those outside. In order to effectively isolate microorganisms from inside the soil aggregates, we washed the surface with electrolyzed NaCl solution, which possesses bactericidal activity, then fractured them with an ultrasonic processor. Gram-positive bacteria with high G+C isolated from the inside of soil aggregates were then compared with those isolated from outside on the basis of their 16S rDNA partial sequences. The results showed that the two sets of isolates differ.
Streptothricins (STs) produced by Streptomyces strains are broad-spectrum antibiotics. All STs consist of a carbamoylated D-gulosamine to which the β-lysine homopolymer (1 to 7 residues) and the amide form of the unusual amino acid streptolidine (streptolidine lactam) are attached. In a previous study, we successfully isolated a novel ST-resistance gene (sttH) from Streptomyces albulus by shotgun cloning with a heterologous host. In vitro analysis of SttH demonstrated that this enzyme catalyzes the hydrolysis of the amide bond of streptolidine lactam, thereby conferring ST resistance. We suggested that the true role of SttH may not be its involvement in resistance against STs; instead, it may catalyze the hydrolysis of naturally occurring cyclic amide compounds in the metabolism of S. albulus. In this study, we therefore constructed an sttH gene-knockout mutant to better understand the true biological role of SttH in S. albulus and to clarify whether or not S. albulus possesses different ST-resistance gene(s). The minimum inhibitory concentration MIC of STs in the sttH gene-knockout mutant was determined to be 6.25 μg/ml, whereas the MIC of STs in the S. albulus wild strain was determined to be >400 μg/ml. This result clearly demonstrated that sttH is the sole gene responsible for ST resistance in the S. albulus strain. Moreover, we developed a transformation system for S. albulus by electroporation in this study.
Type-II polyketide synthases (PKS) are composed of two β-ketoacyl synthase subunits (KSα and KSβ) and one acyl carrier protein, which are involved in the biosynthesis of polyketide molecules. In this study, we investigated the diversity of type-II pks genes in 83 Streptomyces strains listed in the catalogue of the NITE Biological Resource Center (NBRC, http://www.nbrc.nite.go.jp/e/index.html) by determining partial sequences of the KSα genes. As expected, KSα genes were detected in 9 strains that have been reported to produce aromatic-polyketides. More interestingly, novel KSα genes were detected in 55 out of 74 Streptomyces strains so far unknown to produce aromatic antibiotics. The antibiotic-type KSα genes exhibited a high degree of divergence, the pairwise nucleotide similarities ranged from 67 to 95%. The existence of novel cryptic KSα genes in the Streptomyces strains suggest their potential to synthesize new aromatic polyketides.
Larvae of the Japanese horned beetle, Trypoxylus dichotomus septentrionalis (Kono 1931), feed on putrid wood and leaf litter, and are assumed to play important roles in the degradation of polysaccharides produced by broadleaf trees in Japan. Wada et al. showed the presence of strong degrading activities against several polysaccharides, e.g., β-1, 4-xylan, pectin, and β-1,3-glucan, in the contents of the larval gut and an alkaline pH (pH 10) of the contents of the larval midgut. Xylanolytic strains of bacteria, H05TDL, F09TDL, and W02TDL, were isolated, by use of alkaline media containing 0.5% xylan, from the hindgut and feces of T. dichotomus larvae and putrid wood used for feeding of the larva; and these strains seemed to belong to the genus Dietzia by 16S rDNA analysis. The strain H05TDL showed higher xylanolytic activity at alkaline pH than at neutral pH. The cells of this strain were Gram-positive aerobic rods containing short-chain mycolic acids (33–40 carbon atoms) and arabinose and galactose as the major cell-wall sugars, which also support that the strain belongs to the genus Dietzia. The bacterium grew well between pH 6.8 and 9.6 (optimally at pH 8.3), and produced β-1,4-xylanase, which showed its maximum activity at pH 9.4. In the pH range between 8 and 10, the enzyme activity was 70% of the maximum activity or higher, suggesting that the β-1,4-xylanase may have become adapted to the alkaline lumen of the gut.
Rhodococci are robust biocatalysts and they show diverse capabilities with regard to the degradation of xenobiotic compounds including recalcitrant organic compounds such as heterocyclic aromatic compounds and polychlorinated biphenyls (PCBs). Furthermore, these microbes exhibit a broad range of enzymes that can produce valuable chemicals such as intermediates that have a medical importance. These distinct characteristics have encouraged researchers to use rhodococci for industrial, environmental, and pharmaceutical applications. Genome projects of several Rhodococcus species have revealed the presence of large plasmids that encode genes related to their specific abilities. Recent advances in the development of genetic tools along with the rapid progress in genome annotations will pave the way for the efficient cell engineering of rhodococci. Particularly, a variety of expression vectors and transposon mutagenesis systems will act as important tools, and the methods for gene expression analysis will become powerful tools for the discovery of desirable genes in cell engineering.
Staurosporine and rebeccamycin are natural antitumor compounds produced by actinomycetes. Indolocarbazole biosynthetic gene clusters have been cloned from both Streptomyces sp. TP-A0274 and Lechevalieria aerocolonigenes ATCC39243. Staurosporine biosynthetic gene cluster consists of 15 orfs spanning 22 kb, and rebeccamycin biosynthetic gene cluster consists of 10 orfs spanning 16 kb. Gene disruption and bioconversion experiments revealed that indole-3-pyruvic acid, which is derived from tryptophan, is coupled to yield chromopyrrolic acid, and then this dicarboxylic bisindole compound is transformed into the indolocarbazole skeleton via two oxidation steps. Goadsporin is another heterocyclic antibiotic produced by Streptomyces sp. TP-A0584. It is a polypeptide antibiotic containing the thiazole and oxazole rings. The biosynthetic goadsporin gene cluster consists of 10 genes: a structural gene godA and nine genes involved in post-translational modification, immunity, and transcriptional regulation. It is particularly noteworthy that godI, which shows high similarity to the subunit of the “signal recognition particle,” plays an important role in goadsporin immunity. Furthermore, combinatorial biosynthesis was applied to produce the indolocarbazole and goadsporin analogs, and as a result, 14 analogs of indolocarbazole and 4 of goadsporin were produced.