The oxazolomycins (OZMs) are a growing family of antibiotics produced by several Streptomyces species that show diverse and important antibacterial, antitumor, and anti-human immunodeficiency virus activity. Oxazolomycin A is a peptidepolyketide hybrid compound containing a unique spiro-linked ␤-lactone/␥-lactam, a 5-substituted oxazole ring. The oxazolomycin biosynthetic gene cluster (ozm) was identified from Streptomyces albus JA3453 and localized to 79.5-kb DNA, consisting of 20 open reading frames that encode non-ribosomal peptide synthases, polyketide synthases (PKSs), hybrid nonribosomal peptide synthase-PKS, trans-acyltransferases (trans-ATs), enzymes for methoxymalonyl-acyl carrier protein (ACP) synthesis, putative resistance genes, and hypothetical regulation genes. In contrast to classical type I polyketide or fatty acid biosynthases, all 10 PKS modules in the gene cluster lack cognate ATs. Instead, discrete ATs OzmM (with tandem domains OzmM-AT1 and OzmM-AT2) and OzmC were equipped to carry out all of the loading functions of both malonyl-CoA and methoxymalonyl-ACP extender units. Strikingly, only OzmM-AT2 is required for OzmM activity for OZM biosynthesis, whereas OzmM-AT1 seemed to be a cryptic AT domain. The above findings, together with previous results using isotope-labeled precursor feeding assays, are assembled for the OZM biosynthesis model to be proposed. The incorporation of both malonylCoA (by OzmM-AT2) and methoxymalonyl-ACP (by OzmC) extender units seemed to be unprecedented for this class of trans-AT type I PKSs, which might be fruitfully manipulated to create structurally diverse novel compounds.

Oxazolomycin (OZM), a hybrid peptide-polyketide antibiotic, exhibits potent antitumor and antiviral activities. Using degenerate primers to clone genes encoding methoxymalonyl-acyl carrier protein (ACP) biosynthesis as probes, a 135-kb DNA region from Streptomyces albus JA3453 was cloned and found to cover the entire OZM biosynthetic gene cluster. The involvement of the cloned genes in OZM biosynthesis was confirmed by deletion of a 12-kb DNA fragment containing six genes for methoxymalonyl-ACP biosynthesis from the specific region of the chromosome, as well as deletion of the ozmC gene within this region, to generate OZM-nonproducing mutants.

The HAD superfamily protein OzmB from the oxazolomycin biosynthetic pathway is shown to divert the primary metabolite 1,3-diphosphoglycerate into the polyketide biosynthetic pathway as glycerate via loading of a carrier protein. Each of the steps-activation of d-3-phosphoglycerate, dephosphorylation while attached to a cysteine on OzmB, and subsequent transfer of glycerate to the phosphopantetheinyl thiol of an acyl carrier protein-was monitored by nanospray Fourier transform mass spectrometry. This activation of phosphoglycerate represents a general mechanism of diverting glycolytic metabolites into glyceryl-derived polyketides.

The rarest codon in the high G+C genome of Streptomyces coelicolor is TTA, corresponding in mRNA to the UUA codon that is recognized by a developmentally important tRNA encoded by the bldA gene. There are 145 TTA-containing genes in the chromosome of S. coelicolor. Only 42 of these are represented in the genome of Streptomyces avermitilis, among which only 12 have a TTA codon in both species. The TTA codon is less represented in housekeeping genes and orthologous genes, and is more represented in functional-unknown, extrachromosomal or weakly expressed genes. Twenty one TTA-containing chromosomal genes in S. coelicolor were disrupted, including 12 of the 42 genes that are common to both S. avermitillis and S. coelicolor. None of the mutant strains showed any obvious phenotypic differences from the wild-type strain under tested conditions. Possible reasons for this, and the role and evolution of the observed distribution of TTA codons among Streptomyces genes were discussed.

Prostate cancer is a common cause of death, and an important goal is to establish the pathways and functions of causative genes. We isolated RNAs that are differentially expressed in macrodissected prostate cancer samples. This study focused on 1 identified gene, TTLL12, which was predicted to modify tubulins, an established target for tumor therapy. TTLL12 is the most poorly characterized member of a recently discovered 14-member family of proteins that catalyze posttranslational modification of tubulins. We show that human TTLL12 is expressed in the proliferating layer of benign prostate. Expression increases during cancer progression to metastasis. It is highly expressed in many metastatic prostate cancer cell lines. It partially colocalizes with vimentin intermediate filaments and cellular structures containing tubulin, including midbodies, centrosomes, intercellular bridges and the mitotic spindle. Downregulation of TTLL12 affects several posttranslational modifications of tubulin (detyrosination and subsequent deglutamylation and polyglutamylation). Overexpression alters chromosomal ploidy. These results raise the possibility that TTLL12 could contribute to tumorigenesis through effects on the cytoskeleton, tubulin modification and chromosome number stability. This study contributes a step toward developing more selective agents targeting microtubules, an already successful target for tumor therapy.

Ascamycin (ACM) and dealanylascamycin (DACM) are nucleoside antibiotics elaborated by Streptomyces sp. JCM9888. The later shows broad spectrum inhibition activity to various gram-positive and gram-negative bacteria, eukaryotic Trypanosoma and is also toxic to mice, while ascamycin is active against very limited microorganisms, such as Xanthomonas. Both compounds share an unusual 5′-O-sulfonamide moiety which is attached to an adenosine nucleoside. In this paper, we first report on the 30 kb gene cluster (23 genes, acmA to acmW) involved in the biosynthesis of these two antibiotics and a biosynthetic assembly line was proposed. Of them, six genes (AcmABGKIW) are hypothetical genes involved in 5′-O-sulfonamide formation. Two flavin adenine dinucleotide (FAD)-dependent chlorinase genes acmX and acmY were characterized which are significantly remote from acmA-W and postulated to be required for adenine C2-halogenation. Notably gene disruption of acmE resulted in a mutant which could only produce dealanylascamycin but was blocked in its ability to biosynthesize ascamycin, revealing its key role of conversion of dealanylascamycin to ascamycin.

Hematopoiesis takes place in close contact with the marrow microenvironment. Normal progenitors adhere through a variety of receptors to stroma and extracellular matrix components, including fibronectin. Adhesion through integrins to fibronectin may not only serve to anchor progenitors to the microenvironment but also to directly alter the proliferative behavior of normal hematopoietic progenitors. Chronic myelogenous leukemia (CML) is a malignant disease of the hematopoietic stem cell. At the molecular level, CML is characterized by the BCR/ABL gene rearrangement which encodes for the oncoprotein, p210^bcr-abl. Presence of the p210^bcr-abl tyrosine kinase is necessary and sufficient for the malignant transformation of hematopoietic cells. Clinically, CML is characterized by an abnormal, premature release of primitive progenitors and precursors in the blood and by the continuous proliferation of the malignant progenitor population. In vitro, CML progenitors fail to adhere to or be regulated by marrow stroma. Since CML progenitors express similar numbers of integrin adhesion receptors as normal progenitors, functional rather than quantitative differences of these receptors on CML progenitors may be responsible for the abnormal circulation and proliferation of the malignant clone. In this manuscript we will review the role of integrin adhesion receptors present on normal hematopoietic progenitors in the regulation of their proliferation and discuss signal transduction mechanisms that may be responsible for these effects. We will also discuss the integrin defect in CML which may be caused by the presence of the oncoprotein, P210^bcr-abl, and may explain the abnormal trafficking and proliferation observed in CML.

Microbiota play important roles in the internal environment and health of humans, livestock and wild animals. Short-chain fatty acids (SCFAs) and lactate are primary metabolites that can impact the composition and function of human microbiota. According to the well-characterized key synthesis genes, many SCFA- and lactate-producing bacteria have been identified in the gut microbiota. However, unknown genes may also contribute to the formation of SCFAs and lactate. The identification of such genes will provide new engineering targets and new strategies for maintaining a stable structure of beneficial microbiota. In this study, we used Escherichia coli as a model to analyze possible genes related to SCFAs and lactate production besides the well-characterized ones. The functions of nineteen candidate genes were studied by targeted gene deletion and overexpression. Results indicated thioesterase genes such as yciA , tesA , tesB , and menI can contribute to acetate and/or butyrate formation. As for lactate, mgsA and lldD can function in addition to ldh gene. At the same time, the distribution of these functional genes in gut microbiota was investigated. Most bacteria contain the well-studied genes whereas some bacteria contain some of the described unusual ones. The results provide insights and genetic targets for the discovery of new SCFA- and lactate-producing bacteria in gut microbiota.