Modeling and Visualizing Bacterial Colony Purification Without the Use of Bacteria or Laboratory Equipment

This paper describes an activity that simulates bacterial purification without the use of biohazardous materials and provides immediate feedback to students regarding the efficacy of their technique

Modeling and Visualizing Bacterial Colony Purification Without the Use of Bacteria or Laboratory Equipment

Microorganisms typically exist in diverse and heterogeneous communities within their various environmental niches.  The isolation of an individual species from these communities is an essential laboratory skill to study of the properties and behaviors of that organism. To achieve this separation, the “quadrant streak” for single colony purification is often included in undergraduate microbiology laboratory curricula.  To aid student mastery of this technique, I have developed a simulated culture purification activity that allows students to immediately visualize the dilution and separation of an artificial microbial community with the goal of isolating purified colonies.  This tool uses readily available, inexpensive, art supplies to simulate a mixed bacterial culture.  The “mixed culture” consists of craft glitter of at least two distinct colors, held together with water-soluble, highly pigmented watercolor or gouache paint. Students practice aseptic technique by using a paintbrush to mimic an inoculating loop to streak and dilute the culture on a piece of cardstock. Sterilization of the “loop” is simulated by rinsing the brush. Students will immediately self-assess whether they are correctly performing the quadrant streak, rather than having to wait until the next laboratory session for bacteria to grow, which may allow them to master the technique sooner

YtxR, a Conserved LysR-Like Regulator That Induces Expression of Genes Encoding a Putative ADP-Ribosyltransferase Toxin Homologue in Yersinia enterocolitica

Yersinia enterocolitica causes human gastroenteritis, and many isolates have been classified as either “American” or “non-American” strains based on their geographic prevalence and virulence properties. In this study we describe identification of a transcriptional regulator that controls expression of the Y. enterocolitica ytxAB genes. The ytxAB genes have the potential to encode an ADP-ribosylating toxin with similarity to pertussis toxin. However, a ytxAB null mutation did not affect virulence in mice. Nevertheless, the ytxAB genes are conserved in many Y. enterocolitica strains. Interestingly, American and non-American strains have different ytxAB alleles encoding proteins that are only 50 to 60% identical. To obtain further insight into the ytxAB locus, we investigated whether it is regulated as part of a known or novel regulon. Transposon mutagenesis identified a LysR-like regulator, which we designated YtxR. Expression of ytxR from a nonnative promoter increased Φ(ytxA-lacZ) operon fusion expression up to 35-fold. YtxR also activated expression of its own promoter. DNase I footprinting showed that a His(6)-YtxR fusion protein directly interacted with the ytxA and ytxR control regions at similar distances upstream of their probable transcription initiation sites, identified by primer extension. Deletion analysis demonstrated that removal of the regions protected by His(6)-YtxR in vitro eliminated YtxR-dependent induction in vivo. The ytxAB locus is not present in most Yersinia species. In contrast, ytxR is conserved in multiple Yersinia species, as well as in the closely related organisms Photorhabdus luminescens and Photorhabdus asymbiotica. These observations suggest that YtxR may play a conserved role involving regulation of other genes besides ytxAB

YtxR acts as an overriding transcriptional off switch for the Yersinia enterocolitica Ysc-Yop type 3 secretion system.

The Yersinia enterocolitica YtxR protein is a LysR-type transcriptional regulator that induces expression of the ytxAB locus, which encodes a putative ADP-ribosylating toxin. The ytxR and ytxAB genes are not closely linked in the Y. enterocolitica chromosome, and whereas ytxR is present in all sequenced Yersinia spp., the ytxAB locus is not. These observations suggested that there might be other YtxR-regulon members besides ytxAB and prompted us to investigate coregulated genes and gene products by using transcriptional and proteomic approaches. Microarray and reverse transcription-PCR analysis showed that YtxR strongly activates expression of the yts2 locus, which encodes a putative type 2 secretion system, as well as several uncharacterized genes predicted to encode extracytoplasmic proteins. Strikingly, we also discovered that under Ysc-Yop type 3 secretion system-inducing conditions, YtxR prevented the appearance of Yop proteins in the culture supernatant. Microarray and lacZ operon fusion analysis showed that this was due to specific repression of ysc-yop gene expression. YtxR was also able to repress VirF-dependent Phi(yopE-lacZ) and Phi(yopH-lacZ) expression in a strain lacking the virulence plasmid, which suggested a direct repression mechanism. This was supported by DNase I footprinting, which showed that YtxR interacted with the yopE and yopH control regions. Therefore, YtxR is a newly identified regulator of the ysc-yop genes that can act as an overriding off switch for this critical virulence system

Proceedings of the CUNY Games Conference 9.0

The CUNY Games Network is an organization dedicated to encouraging research, scholarship and teaching in the developing field of games-based learning. We connect educators from every campus and discipline at CUNY and beyond who are interested in digital and non-digital games, simulations, and other forms of interactive teaching and inquiry-based learning

Malignant Tumors and Forensics – Dilemmas and Proposals

Aim To evaluate the effect of genetic instability and degradation in archived histology samples from cancerous tumors and to investigate the validity of short tandem repeat (STR) typing of these samples and its potential effect on human identification. Methods Two hundred and twenty eight slides of archival pathology tissues from 13 different types of malignant tumors were compared with healthy tissues from the same individuals. DNA analysis was performed using standard techniques for forensic STR analysis, PowerPlex®16 and Identifiler® on 2 distinct sample sets. Genetic instability was assessed by comparing reference tissues with cancerous tissues derived from the same individual. Loss of heterozygosity, a ≥50% reduction in heterozygosity ratio between healthy and diseased samples, and microsatellite instability, the presence of an additional allele not present in reference tissue, were assessed. The quality of profiles obtained with respect to completeness among the archived samples and degradation using the 2 platforms were also compared. Results Profiles obtained using the Identifiler® system were generally more complete, but showed 3-fold higher levels of instability (86%) than those obtained using PowerPlex ® 16 (27%). Instances of genetic instability were distributed throughout all loci in both multiplex STR systems. Conclusion After having compared 2 widely used forensic chemistries, we suggest individual validation of each kit for use with samples likely to exhibit instability combined with fixation induced degradation or artifact. A “one size fits all” approach for interpretation of these samples among commercially available multiplexes is not recommended

Malignant Tumors and Forensics – Dilemmas and Proposals

Aim To evaluate the effect of genetic instability and degradation in archived histology samples from cancerous tumors and to investigate the validity of short tandem repeat (STR) typing of these samples and its potential effect on human identification. Methods Two hundred and twenty eight slides of archival pathology tissues from 13 different types of malignant tumors were compared with healthy tissues from the same individuals. DNA analysis was performed using standard techniques for forensic STR analysis, PowerPlex®16 and Identifiler® on 2 distinct sample sets. Genetic instability was assessed by comparing reference tissues with cancerous tissues derived from the same individual. Loss of heterozygosity, a ≥50% reduction in heterozygosity ratio between healthy and diseased samples, and microsatellite instability, the presence of an additional allele not present in reference tissue, were assessed. The quality of profiles obtained with respect to completeness among the archived samples and degradation using the 2 platforms were also compared. Results Profiles obtained using the Identifiler® system were generally more complete, but showed 3-fold higher levels of instability (86%) than those obtained using PowerPlex ® 16 (27%). Instances of genetic instability were distributed throughout all loci in both multiplex STR systems. Conclusion After having compared 2 widely used forensic chemistries, we suggest individual validation of each kit for use with samples likely to exhibit instability combined with fixation induced degradation or artifact. A “one size fits all” approach for interpretation of these samples among commercially available multiplexes is not recommended

Co-ordinated regulation of two divergent promoters through higher-order complex formation by the LysR-type regulator ThnR

15 páginas, 8 figuras, 2 tablas.The genes required for tetralin biodegradation by Sphingomonas macrogolitabida strain TFA are clustered in two divergent and closely linked operons. ThnR, a LysR-type regulator, activates transcription from each operon in response to tetralin. The regulatory thnR gene is co-transcribed with the catabolic genes thnC, thnA3 and thnA4, resulting in positive autoregulation. ThnR binds with different affinity to two primary binding sites, designated B and C, in the intervening region between the two operons and makes additional contact with secondary sites that extend towards the promoters. In addition, ThnR may interact with itself when bound to each site via the formation of a DNA loop, as evidenced by the distortion of the DNA between the primary binding sites and the elimination of the higher-order complexes following the introduction of a half-turn of the DNA helix between the primary binding sites. Transcription from each promoter is not fully independent since mutations in each binding site affected transcription from both promoters. Based on these results, we propose a model of transcription activation that involves the formation of a complex structure by interactions between ThnR molecules bound to distant binding sites and favours transcription from one promoter to the detriment of the other.This work was supported by the Spanish Ministry of Science and Innovation, Grants BIO2008-01805 and CSD2007-00005, and by the Andalusian government, Grants P05-CVI-131 and P07-CVI-2518.Peer reviewe