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Xylose is an abundant, inexpensive and readily available carbohydrate common in minimally processed feedstocks such as seaweed and algae. While a wide variety of marine microbes have evolved to utilize seaweed and algae, only a few currently have the requisite characteristics and genetic engineering tools necessary to entertain the use of these underutilized feedstocks. The rapidly growing Gram‐negative halophilic bacterium Vibrio natriegens is one such chassis. In this study, we engineered and tested xylose induction in V. natriegens as a tool for scalable bioproduction applications. First, we created a sensing construct based on the xylose operon from Escherichia coli MG1665 and measured its activity using a fluorescent reporter and identified that cellular import plays a key role in induction strength and that expression required the XylR transcription factor. Next, we identified that select deletions of the promoter region enhance gene expression, limiting the effect of carbohydrate repression when xylose is used as an inducer in the presence of industrially relevant carbon sources. Lastly, we used the optimized constructs to produce the biopolymer melanin using seawater mimetic media. One of these formulations utilized a nori‐based seaweed extract as an inducer and demonstrated melanin yields comparable to previously optimized methods using a more traditional and costly inducer. Together, the results demonstrate that engineering xylose induction in V. natriegens can provide an effective and lower cost option for timed biosynthesis in scalable biomanufacturing applications using renewable feedstocks. [ABSTRACT FROM AUTHOR]

The discovery of non-coding RNA (ncRNA) has been mainly limited to laboratory model systems and human pathogenic bacteria. In this study, we begin to explore the ncRNA diversity in four recently sequenced environmental Vibrio species (Vibrio alginolyficus 40B, Vibrio communis 1DA3, Vibrio mimicus VM573 and Vibrio campbellii BAA-1116) by performing in silico searches using Infernal and Rfam for the identification of putative ncRNA-encoding genes. This search method resulted in the identification of 31-38 putative ncRNA genes per species and the total ncRNA catalogue spanned an assortment of regulatory mechanisms (riboswitches, cis-encoded ncRNAs, trans-encoded ncRNAs, modulators of protein activity, ribonucleoproteins, transcription termination ncRNAs and unknown). We chose to experimentally validate the identifications for V. campbellii BAA-1116 using a microarray-based expression profiling strategy. Transcript hybridization to tiled probes targeting annotated V. campbellii BAA-1116 intergenic regions revealed that 21 of the 38 predicted ncRNA genes were expressed in mid-exponential-phase cultures grown in nutrient-rich medium. The microarray findings were confirmed by testing a subset of three highly expressed (6S, tmRNA and TPP-2) and three moderately expressed (CsrB, GcvB and purine) ncRNAs via reverse transcription PCR. Our findings provide new information on the diversity of ncRNA in environmental vibrios while simultaneously promoting a more accurate annotation of genomic intergenic regions. DOI 10.1099/mic.0.039149-0

Despite the success of the adenovirus vaccine administered to US military trainees, acute respiratory disease (ARD) surveillance still detected breakthrough infections (respiratory illnesses associated with the adenovirus serotypes specifically targeted [...]

An enhancement in the fluorescent signal of the carbocyanine dye Cy5 by using an engineered virus as a scaffold to attach >40 Cy5 reporter molecules at fixed location on the viral capsid are presented. When compared with the most often used detection methods in a microarray based genotyping assay for vibrio cholerae O139, these viral nanoparticles markedly increased assay sensitivity, thus demonstrating their applicability for existing DNA microarray protocols.

Identification of genetic variations of influenza viruses is essential for epidemic and pandemic outbreak surveillance and determination of vaccine strain selection. In this study, we combined a random amplification strategy [...]

The morbidity and mortality associated with Vibrio-mediated waterborne diseases necessitates the development of sensitive detection technologies that are able to elucidate the identity, potential pathogenicity, susceptibility, and viability of contaminating bacteria in a timely manner. For this purpose, we have designed a single multiplex PCR assay to simultaneously amplify 95 diagnostic regions (encompassing species/serogroup-specific, antimicrobial resistance, and known toxin markers) and combined it with a long oligonucleotide microarray to create a platform capable of rapidly detecting and discriminating the major human pathogenic species from the genus Vibrio: V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus. We were able to validate this strategy by testing 100 geographically and temporally distributed isolates and observed an excellent concordance between species- and serotype-level microarray-based identification and traditional typing methods. In addition to accurate identification, the microarray simultaneously provided evidence of antibiotic resistance genes and mobile genetic elements, such as sulfamethoxazole-trimethoprim constins and class I integrons, and common toxin (ctxAB, rtxA, hap, hlyA, tl, tdh, trh, vvhA, vlly, and vmhA) and pathogenicity (tcpA, type III secretion system) genes that are associated with pathogenic Vibrio. The versatility of this method was further underscored by its ability to detect the expression of known toxin and virulence genes from potentially harmful viable but nonculturable organisms. The results suggest that this molecular identification method provides rapid and definitive information that would be of value in epidemiological, environmental, and health risk assessment surveillance. pathogen detection | molecular diagnostics | cholera

Four front-end based amplification strategies such as random primed, isothermal klenow fragment-based, 29 DNA polymerase-based, and multiplex PCR are investigated. The results presented underscore the feasibility of using random amplification approaches and begin to systematically address the versatility if these approaches for unbiased pathogen detection from environmental sources.