High-resolution mapping of plasmid transcriptomes in different host bacteria

<p>Abstract</p> <p>Background</p> <p>Plasmids are extrachromosomal elements that replicate autonomously, and many can be transmitted between bacterial cells through conjugation. Although the transcription pattern of genes on a plasmid can be altered by a change in host background, the expression range of plasmid genes that will result in phenotypic variation has not been quantitatively investigated.</p> <p>Results</p> <p>Using a microarray with evenly tiled probes at a density of 9 bp, we mapped and quantified the transcripts of the carbazole catabolic plasmid pCAR1 in its original host <it>Pseudomonas resinovorans </it>CA10 and the transconjugant <it>P</it>. <it>putida </it>KT2440(pCAR1) during growth on either carbazole or succinate as the sole carbon source. We identified the operons in pCAR1, which consisted of nearly identical transcription units despite the difference in host background during growth on the same carbon source. In accordance with previous studies, the catabolic operons for carbazole degradation were upregulated during growth on carbazole in both hosts. However, our tiling array results also showed that several operons flanking the transfer gene cluster were transcribed at significantly higher levels in the transconjugant than in the original host. The number of transcripts and the positions of the transcription start sites agreed with our quantitative RT-PCR and primer extension results.</p> <p>Conclusion</p> <p>Our tiling array results indicate that the levels of transcription for the operons on a plasmid can vary by host background. High-resolution mapping using an unbiased tiling array is a valuable tool for the simultaneous identification and quantification of prokaryotic transcriptomes including polycistronic operons and non-coding RNAs.</p

Distribution of Genes Encoding Nucleoid-Associated Protein Homologs in Plasmids

Bacterial nucleoid-associated proteins (NAPs) form nucleoprotein complexes and influence the expression of genes. Recent studies have shown that some plasmids carry genes encoding NAP homologs, which play important roles in transcriptional regulation networks between plasmids and host chromosomes. In this study, we determined the distributions of the well-known NAPs Fis, H-NS, HU, IHF, and Lrp and the newly found NAPs MvaT and NdpA among the whole-sequenced 1382 plasmids found in Gram-negative bacteria. Comparisons between NAP distributions and plasmid features (size, G+C content, and putative transferability) were also performed. We found that larger plasmids frequently have NAP gene homologs. Plasmids with H-NS gene homologs had less G+C content. It should be noted that plasmids with the NAP gene homolog also carried the relaxase gene involved in the conjugative transfer of plasmids more frequently than did those without the NAP gene homolog, implying that plasmid-encoded NAP homologs positively contribute to transmissible plasmids

Characterization of bacterial community structure in a hydrocarbon-contaminated tropical African soil

The bacterial community structure in a hydrocarbon-contaminated Mechanical Engineering Workshop (MWO) soil was deciphered using 16S rRNA gene clone library analysis. Four hundred and thirty-seven clones cutting across 13 bacterial phyla were recovered from the soil. The representative bacterial phyla identified from MWO soil are Proteobacteria, Bacteroidetes, Chloroflexi, Acidobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Planctomycetes, Ignavibacteriae, Spirochaetes, Chlamydiae, Candidatus Saccharibacteria and Parcubacteria. Proteobacteria is preponderant in the contaminated soil (51.2%) with all classes except Epsilonproteobacteria duly represented. Rarefaction analysis indicates 42%, 52% and 77% of the clone library is covered at the species, genus and family/class delineations with Shannon diversity (H′) and Chao1 richness indices of 5.59 and 1126, respectively. A sizeable number of bacterial phylotypes in the clone library shared high similarities with strains previously described to be involved in hydrocarbon biodegradation. Novel uncultured genera were identified that have not been previously reported from tropical African soil to be associated with natural attenuation of hydrocarbon pollutants. This study establishes the involvement of a wide array of physiologically diverse bacterial groups in natural attenuation of hydrocarbon pollutants in soil

Low-cost gel-filled microwell array device for screening marine microbial consortium

In order to exploit the microbes present in the environment for their beneficial resources, effective selection and isolation of microbes from environmental samples is essential. In this study, we fabricated a gel-filled microwell array device using resin for microbial culture. The device has an integrated sealing mechanism that enables high-density isolation based on the culture of microorganisms; the device is easily manageable, facilitating observation using bright-field microscopy. This low-cost device made from polymethyl methacrylate (PMMA)/polyethylene terephthalate (PET) has 900 microwells (600 μm × 600 μm × 700 μm) filled with a microbial culture gel medium in glass slide-sized plates. It also has grooves for maintaining the moisture content in the micro-gel. The partition wall between the wells has a highly hydrophobic coating to inhibit microbial migration to neighboring wells and to prevent exchange of liquid substances. After being hermetically sealed, the device can maintain moisture in the agarose gels for 7 days. In the bacterial culture experiment using this device, environmental bacteria were isolated and cultured in individual wells after 3 days. Moreover, the isolated bacteria were then picked up from wells and re-cultured. This device is effective for the first screening of microorganisms from marine environmental samples

Biocide Resistance and Transmission of Clostridium difficile Spores Spiked onto Clinical Surfaces from an American Health Care Facility

Clostridium difficile is the primary cause of antibiotic-associated diarrhea globally. In unfavourable environments the organism produces highly resistant spores which can survive microbicidal insult. Our previous research determined the ability of C. difficile spores to adhere to clinical surfaces; finding that spores had marked different hydrophobic properties and adherence ability. Investigation into the effect of the microbicide sodium dichloroisocyanurate on C. difficile spore transmission revealed that sub-lethal concentrations increased spore adherence without reducing viability. The present study examined the ability of spores to transmit across clinical surfaces and their response to an in-use disinfection concentration of 1,000-ppm of chlorine-releasing agent sodium dichloroisocyanurate. In an effort to understand if these surfaces contribute to nosocomial spore transmission, surgical isolation gowns, hospital-grade stainless steel and floor vinyl were spiked with 1 × 106 spores/ml of two types of C. difficile spore preparations: crude spores and purified spores. The hydrophobicity of each spore type versus clinical surface was examined via plate transfer assay and scanning electron microscopy. The experiment was repeated and spiked clinical surfaces were exposed to 1,000-ppm sodium dichloroisocyanurate at the recommended 10-min contact time. Results revealed that the hydrophobicity and structure of clinical surfaces can influence spore transmission and that outer spore surface structures may play a part in spore adhesion. Spores remained viable on clinical surfaces after microbicide exposure at the recommended disinfection concentration demonstrating ineffectual sporicidal action. This study showed that C. difficile spores can transmit and survive between varying clinical surfaces despite appropriate use of microbicides. IMPORTANCE Clostridium difficile is a healthcare-acquired organism and the causative agent of antibiotic-associated diarrhoea. Its spores are implicated in faecal to oral transmission from contaminated surfaces in the healthcare environment due to their adherent nature. Contaminated surfaces are cleaned using high-strength chemicals to remove and kill the spores; however, despite appropriate infection control measures, there is still high incidence of C. difficile infection in patients in the US. Our research examined the effect of a high-strength biocide on spores of C. difficile which had been spiked onto a range of clinically relevant surfaces including isolation gowns, stainless steel and floor vinyl. This study found that C. difficile spores were able to survive exposure to appropriate concentrations of biocide; highlighting the need to examine the effectiveness of infection control measures to prevent spore transmission, and consideration of the prevalence of biocide resistance when decontaminating healthcare surfaces.</jats:p

Diversity of Nonribosomal Peptide Synthetase Genes in the AnticancerProducing Actinomycetes Isolated from Marine Sediment in Indonesia

Marine actinomycetes is a group of bacteria that is highly potential in producing novel bioactive compound. It has unique characteristics and is different from other terrestrial ones. Extreme environmental condition is suspected to lead marine actinomycetes produce different types of bioactive compound found previously. The aim of this study was to explore the presence and diversity of NRPS genes in 14 anticancer-producing actinomycetes isolated from marine sediment in Indonesia. PCR amplification and restriction fragment analysis of NRPS genes with HaeIII from 14 marine actinomycetes were done to assess the diversity of NRPS genes. Genome mining of one species of marine actinomycetes (strain GMY01) also was employed towards this goal. The result showed that NRPS gene sequence diversity in 14 marine actinomycetes could be divided into 4 groups based on NRPS gene restriction patterns. Analysis of 16S rRNA gene sequences of representatives from each group showed that all isolates belong to genus of Streptomyces. Genome mining result showed that strain GMY01 harboring 10 different NRPS gene clusters that encode secondary metabolites, as pure NRPS or hybrid between NRPS and other compounds. These results indicated that marine actinomycetes having a high potential to be developed as source of anticancer drugs development