Remedial Treatment of Corroded Iron Objects by Environmental Aeromonas Isolates.

Using bacteria to transform reactive corrosion products into stable compounds represents an alternative to traditional methods employed in iron conservation. Two environmental Aeromonas strains (CA23 and CU5) were used to transform ferric iron corrosion products (goethite and lepidocrocite) into stable ferrous iron-bearing minerals (vivianite and siderite). A genomic and transcriptomic approach was used to analyze the metabolic traits of these strains and to evaluate their pathogenic potential. Although genes involved in solid-phase iron reduction were identified, key genes present in other environmental iron-reducing species are missing from the genome of CU5. Several pathogenicity factors were identified in the genomes of both strains, but none of these was expressed under iron reduction conditions. Additional in vivo tests showed hemolytic and cytotoxic activities for strain CA23 but not for strain CU5. Both strains were easily inactivated using ethanol and heat. Nonetheless, given a lesser potential for a pathogenic lifestyle, CU5 is the most promising candidate for the development of a bio-based iron conservation method stabilizing iron corrosion. Based on all the results, a prototype treatment was established using archaeological items. On those, the conversion of reactive corrosion products and the formation of a homogenous layer of biogenic iron minerals were achieved. This study shows how naturally occurring microorganisms and their metabolic capabilities can be used to develop bio-inspired solutions to the problem of metal corrosion.IMPORTANCE Microbiology can greatly help in the quest for a sustainable solution to the problem of iron corrosion, which causes important economic losses in a wide range of fields, including the protection of cultural heritage and building materials. Using bacteria to transform reactive and unstable corrosion products into more-stable compounds represents a promising approach. The overall aim of this study was to develop a method for the conservation and restoration of corroded iron items, starting from the isolation of iron-reducing bacteria from natural environments. This resulted in the identification of a suitable candidate (Aeromonas sp. strain CU5) that mediates the formation of desirable minerals at the surfaces of the objects. This led to the proof of concept of an application method on real objects.

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome.

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation allows culturing of a diverse microbial community and provides hundreds to thousands of genetically identical cells as input for an MDA reaction. We demonstrate the utility of this approach by comparing sequencing results of gel microdroplets and single cells following MDA. Bias is reduced in the MDA reaction and genome sequencing, and assembly is greatly improved when using gel microdroplets. We acquired multiple near-complete genomes for two bacterial species from human oral and stool microbiome samples. A significant amount of genome diversity, including single nucleotide polymorphisms and genome recombination, is discovered. Gel microdroplets offer a powerful and high-throughput technology for assembling whole genomes from complex samples and for probing the pan-genome of naturally occurring populations.

Investigating intestinal permeability and gut microbiota roles in acute coronary syndrome patients.

Background: Acute Coronary Syndrome (ACS) is a leading cause of morbidity and mortality. Perturbed gut- microbiota (dysbiosis) and increased intestinal permeability (leaky-gut) with translocation of bacterial antigens, play critical role in obesity and metabolic syndrome, which are also major ACS risk factors. Additionally, Trimethylamine-N-Oxide (TMAO), a metabolite produced by phylum Proteobacteria in gut is implicated in developing ACS. As Proteobacteria is a major source of translocated antigen lipopolysaccharides (LPS), we hypothesized that ACS patients have leaky-gut condition characterized by dysbiosis with increased Proteobacteria, leading to elevated blood levels of TMAO and LPS. Methods: In a pilot case-control study, we enrolled 19 ACS patients (within 72-h of cardiac events) and 19 healthy-controls. Gut barrier function was determined using lactulose-to-mannitol urinary excretion ratio (L/M ratio). Stool microbiome composition was examined using16S sequencing and predictive functional analysis for LPS biosynthesis pathway by PICRUSt tool. Serum TMAO and LPS levels were measured. Results: ACS patients had increased Gammaproteobacteria compared to controls:1.8 ±3.0 vs. 0.2 ±0.4% (P =0.04). Though Proteobacteria level was increased but not statistically significant: 4.1 ±3.8 vs. 2.1 ±1.7% (P =0.056). L/M-ratio was three times higher in ACS patients; 0.06 ±0.07 vs 0.023 ±0.02, (P =0.014). Surprisingly, there was no difference in the mean serum LPS or TMAO levels. However, PICRUSt analysis indicated increased Proteobacteria population increasingly contributed to LPS biosynthesis in ACS patients only. Conclusions: ACS patients likely to have leaky-gut and perturbed gut microbiota. Further studies are required to precisely define the role of dysbiosis in ACS.

Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer.

We report herein the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formation and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.

Genome Sequence of Aeribacillus pallidus Strain GS3372, an Endospore-Forming Bacterium Isolated in a Deep Geothermal Reservoir.

The genome of strain GS3372 is the first publicly available strain of Aeribacillus pallidus. This endospore-forming thermophilic strain was isolated from a deep geothermal reservoir. The availability of this genome can contribute to the clarification of the taxonomy of the closely related Anoxybacillus, Geobacillus, and Aeribacillus genera.

Genome Sequence of Bacillus alveayuensis Strain 24KAM51, a Halotolerant Thermophile Isolated from a Hydrothermal Vent.

Bacillus alveayuensis strain 24KAM51 was isolated from a marine hydrothermal vent in Milos, Greece. Its genome depicts interesting features of halotolerance and resistance to heavy metals.

Genome Sequence of Anoxybacillus geothermalis Strain GSsed3, a Novel Thermophilic Endospore-Forming Species.

Anoxybacillus geothermalis strain GSsed3 is an endospore-forming thermophilic bacterium isolated from filter deposits in a geothermal site. This novel species has a larger genome size (7.2 Mb) than that of any other Anoxybacillus species, and it possesses genes that support its phenotypic metabolic characterization and suggest an intriguing link to metals.

Draft Genome Sequence of Thauera sp. Strain SWB20, Isolated from a Singapore Wastewater Treatment Facility Using Gel Microdroplets.

We report here the genome sequence of Thauera sp. strain SWB20, isolated from a Singaporean wastewater treatment facility using gel microdroplets (GMDs) and single-cell genomics (SCG). This approach provided a single clonal microcolony that was sufficient to obtain a 4.9-Mbp genome assembly of an ecologically relevant Thauera species.

Facile, high quality sequencing of bacterial genomes from small amounts of DNA.

Sequencing bacterial genomes has traditionally required large amounts of genomic DNA (~1 µg). There have been few studies to determine the effects of the input DNA amount or library preparation method on the quality of sequencing data. Several new commercially available library preparation methods enable shotgun sequencing from as little as 1 ng of input DNA. In this study, we evaluated the NEBNext Ultra library preparation reagents for sequencing bacterial genomes. We have evaluated the utility of NEBNext Ultra for resequencing and de novo assembly of four bacterial genomes and compared its performance with the TruSeq library preparation kit. The NEBNext Ultra reagents enable high quality resequencing and de novo assembly of a variety of bacterial genomes when using 100 ng of input genomic DNA. For the two most challenging genomes (Burkholderia spp.), which have the highest GC content and are the longest, we also show that the quality of both resequencing and de novo assembly is not decreased when only 10 ng of input genomic DNA is used.

Artificial polyploidy improves bacterial single cell genome recovery.

BACKGROUND: Single cell genomics (SCG) is a combination of methods whose goal is to decipher the complete genomic sequence from a single cell and has been applied mostly to organisms with smaller genomes, such as bacteria and archaea. Prior single cell studies showed that a significant portion of a genome could be obtained. However, breakages of genomic DNA and amplification bias have made it very challenging to acquire a complete genome with single cells. We investigated an artificial method to induce polyploidy in Bacillus subtilis ATCC 6633 by blocking cell division and have shown that we can significantly improve the performance of genomic sequencing from a single cell. METHODOLOGY/PRINCIPAL FINDINGS: We inhibited the bacterial cytoskeleton protein FtsZ in B.subtilis with an FtsZ-inhibiting compound, PC190723, resulting in larger undivided single cells with multiple copies of its genome. qPCR assays of these larger, sorted cells showed higher DNA content, have less amplification bias, and greater genomic recovery than untreated cells. SIGNIFICANCE: The method presented here shows the potential to obtain a nearly complete genome sequence from a single bacterial cell. With millions of uncultured bacterial species in nature, this method holds tremendous promise to provide insight into the genomic novelty of yet-to-be discovered species, and given the temporary effects of artificial polyploidy coupled with the ability to sort and distinguish differences in cell size and genomic DNA content, may allow recovery of specific organisms in addition to their genomes.

Genomics for key players in the N cycle from guinea pigs to the next frontier.

While sequencing methods were available in the late 1970s, it was not until the human genome project and a significant influx of funds for such research that this technology became high throughput. The fields of microbiology and microbial ecology, among many others, have been tremendously impacted over the years, to such an extent that the determination of complete microbial genome sequences is now commonplace. Given the lower costs of next-generation sequencing platforms, even small laboratories from around the world will be able to generate millions of base pairs of data, equivalent to entire genomes worth of sequence information. With this prospect just around the corner, it is timely to provide an overview of the genomics process: from sample preparation to some of the analytical methods used to gain functional knowledge from sequence information.

The complete genome sequence of Bacillus thuringiensis Al Hakam.

Bacillus thuringiensis is an insect pathogen that is widely used as a biopesticide (E. Schnepf, N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D. R. Zeigler, and D. H. Dean, Microbiol. Mol. Biol. Rev. 62:775-806, 1998). Here we report the finished, annotated genome sequence of B. thuringiensis Al Hakam, which was collected in Iraq by the United Nations Special Commission (L. Radnedge, P. Agron, K. Hill, P. Jackson, L. Ticknor, P. Keim, and G. Andersen, Appl. Environ. Microbiol. 69:2755-2764, 2003).

Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp. konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.