The filamentous bacterium Streptomyces coelicolor differentiates by forming specialized, spore-bearing aerial hyphae that grow into the air. Using microarrays, we identified genes that are down-regulated in a mutant unable to erect aerial hyphae. Through this route, we identified a previously unknown layer of aerial mycelium surface proteins (the "chaplins"). The chaplins share a hydrophobic domain of ∼40 residues (the "chaplin domain"), and all have a secretion signal. The five short chaplins (ChpD,E,F,G,H) have one chaplin domain, whereas the three long chaplins (ChpA,B,C) have two chaplin domains and a C-terminal "sorting signal" that targets them for covalent attachment to the cell wall by sortase enzyme. Expression of the two chaplin genes examined (chpE, chpH) depended on aerial hyphae formation but not sporulation, and egfp fusions showed their expression localized to aerial structures. Mass spectrometry of cell wall extracts confirmed that the short chaplins localized to the cell surface. Deletion of chaplin genes caused severe delays in aerial hyphae formation, a phenotype rescued by exogenous application of chaplin proteins. These observations implicate the chaplins in aerial mycelium formation, and suggest that coating of the envelope by the chaplins is required for aerial hyphae to grow out of the aqueous environment of the substrate mycelium into the air.

SummaryA complex programme of regulation governs gene expression during development of the morphologically and biochemically complex eubacterial genus Streptomyces . Earlier work has suggested a model in which 'higher level' pleiotropic regulators activate 'pathway-specific' regulators located within chromosomal gene clusters encoding biosynthesis of individual antibiotics. We used mutational analysis and adventitious overexpression of key Streptomyces coelicolor regulators to investigate functional interactions among them. We report here that clustersituated regulators (CSRs) thought to be pathwayspecific can also control other antibiotic biosynthetic gene clusters, and thus have pleiotropic actions. Surprisingly, we also find that CSRs exhibit growthphase-dependent control over afsR2/afsS , a 'higher level' pleiotropic regulatory locus not located within any of the chromosomal gene clusters it targets, and further demonstrate that cross-regulation by CSRs is modulated globally and differentially during the S. coelicolor growth cycle by the RNaseIII homologue AbsB. Our results, which reveal a network of functional interactions among regulators that govern production of antibiotics and other secondary metabolites in S. coelicolor , suggest that revision of the currently prevalent view of higher-level versus pathway-specific regulation of secondary metabolism in Streptomyces species is warranted.

The black tiger shrimp (Penaeus monodon) is a marine crustacean of economic importance in the world market. To ensure sustainability of the shrimp industry, production capacity and disease outbreak prevention must be improved. Understanding healthy microbial balance inside the shrimp intestine can provide an initial step toward better farming practice and probiotic applications. In this study, we employed a barcode pyrosequencing analysis of V3-4 regions of 16S rRNA genes to examine intestinal bacteria communities in wild-caught and domesticated P. monodon broodstock. Shrimp faeces were removed from intestines prior to further analysis in attempt to identify mucosal bacterial population. Five phyla, Actinobacteria, Fusobacteria, Proteobacteria, Firmicutes and Bacteroidetes, were found in all shrimp from both wild and domesticated environments. The operational taxonomic unit (OTU) was assigned at 97% sequence identity, and our pyrosequencing results identified 18 OTUs commonly found in both groups. Sequences of the shared OTUs were similar to bacteria in three phyla, namely i) Proteobacteria (Vibrio, Photobacterium, Novosphingobium, Pseudomonas, Sphingomonas and Undibacterium), ii) Firmicutes (Fusibacter), and iii) Bacteroidetes (Cloacibacterium). The shared bacterial members in P. monodon from two different habitats provide evidence that the internal environments within the host shrimp also exerts selective pressure on bacterial members. Intestinal bacterial profiles were compared using denaturing gradient gel electrophoresis (DGGE). The sequences from DGGE bands were similar to those of Vibrio and Photobacterium in all shrimp, consistent with pyrosequencing results. This work provides the first comprehensive report on bacterial populations in the intestine of adult black tiger shrimp and reveals some similar bacterial members between the intestine of wild-caught and domesticated shrimp.

SummaryThe differentiating bacterium Streptomyces coelicolor harbours some 66 sigma factors, which support its complex life cycle.

Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities.

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The potentially important roles of intestinal bacteria on immune response, disease resistance, and nutrition for the black tiger shrimp Penaeus monodon have been increasingly investigated. However, so far, little is known about the intestinal bacterial community of the shrimp in the commercial aquaculture settings. In this study, the intestinal bacterial communities of juvenile P. monodon (70 individuals) from eight commercial farms in Thailand were examined using 16S rDNA PCR-DGGE, and seven 16S rDNA clone libraries from representative DGGE profiles were constructed. Bacteria in the γ-Proteobacteria class were the only common bacteria group found in the intestinal tracts of shrimp from all farms. The dominant bacterial genera in the intestinal population of each shrimp varied among different farms, and these genera were Vibrio, Photobacterium, Aeromonas, or Propionigenium (phylum Fusobacteria). Other commonly found genera included Actinomyces, Anaerobaculum, Halospirulina, Pseudomonas, Mycoplasma, and Shewanella. Twelve phyla of bacteria including Proteobacteria, Firmicutes, Fusobacteria, Actinobacteria, Cyanobacteria, Tenericutes, Deinococcus-Thermus, Planctomycetes, Spirochaetes, Synergistetes, Thermotogae, and Verrucomicrobia were represented in the sequences. Additionally, strictly anaerobic bacteria such as Propionigenium and Fusibacter were found. These intestinal bacterial communities varied significantly among different commercial farms and were distinct from their rearing water. The results provide descriptive structures of the intestinal bacterial communities of P. monodon in commercial farms, which can further be applied to areas of research on the immunity, disease resistance, and nutrition of shrimp to improve aquaculture of the black tiger shrimp.

Eyestalk ablation is commonly practiced in crustacean to induce ovarian maturation in captivity. The molecular mechanism of the ablation has not been well understood, preventing a search for alternative measures to induce ovarian maturation in aquaculture. This is the first study to employ cDNA microarray to examine effects of eyestalk ablation at the transcriptomic level and pathway mapping analysis to identify potentially affected biological pathways in the black tiger shrimp (Penaeus monodon). Microarray analysis comparing between gene expression levels of ovaries from eyestalk-intact and eyestalk-ablated brooders revealed 682 differentially expressed transcripts. Based on Hierarchical clustering of gene expression patterns, Gene Ontology annotation, and relevant functions of these differentially expressed genes, several gene groups were further examined by pathway mapping analysis. Reverse-transcriptase quantitative PCR analysis for some representative transcripts confirmed microarray data. Known reproductive genes involved in vitellogenesis were dramatically increased during the ablation. Besides these transcripts expected to be induced by the ablation, transcripts whose functions involved in electron transfer mechanism, immune responses and calcium signal transduction were significantly altered following the ablation. Pathway mapping analysis revealed that the activation of gonadotropin-releasing hormone signaling, calcium signaling, and progesterone-mediated oocyte maturation pathways were putatively crucial to ovarian maturation induced by the ablation. These findings shed light on several possible molecular mechanisms of the eyestalk ablation effect and allow more focused investigation for an ultimate goal of finding alternative methods to replace the undesirable practice of the eyestalk ablation in the future.