Mining transcriptome data: Utilization of environmentally regulated promoters for protein expression and purification in Clostridium perfringens.

Clostridium perfringens is a Gram-positive pathogen with low GC content. To identify genes that are transcribed at higher levels when the bacteria grow on a surface, we used RNA-seq in a previous study to measure global transcript levels of cells grown in three types of media on both plates and in liquid culture. We found the arcABDC-argR operon is induced >1000-fold when the cells were grown on plates than in liquid brain heart infusion (BHI). In addition, the pyrBICFZDE operon was transcribed >1000-fold higher in liquid BHI than on plates. Biochemical analysis of C. perfringens proteins is usually accomplished by cloning and expressing the relevant genes in Escherichia coli, a Gram-negative bacterium. Here we utilize both the arcA and pyrB promoters to express and purify proteins from C. perfringens plate and liquid-grown cultures, respectively. Three mg of the His-tagged cytoplasmic protein PilM were obtained when the pilM gene was expressed in cells grown on 10 BHI plates using the arcA promoter. Using the pyrB promoter, 0.85 mg of the C. perfringens His-tagged secreted toxin collagenase was purified from the culture supernatant of 500 ml of cells grown in liquid BHI. In the process of constructing clones, we found we can transform C. perfringens strain HN13 directly with DNA from an in vitro ligation mix, bypassing E. coli. These environmentally regulated promoters can be used to express clostridial or other low GC content genes for protein purification without the addition of an inducer molecule.

Drug resistance in diploid yeast is acquired through dominant alleles, haploinsufficiency, gene duplication and aneuploidy.

Previous studies of adaptation to the glucose analog, 2-deoxyglucose, by Saccharomyces cerevisiae have utilized haploid cells. In this study, diploid cells were used in the hope of identifying the distinct genetic mechanisms used by diploid cells to acquire drug resistance. While haploid cells acquire resistance to 2-deoxyglucose primarily through recessive alleles in specific genes, diploid cells acquire resistance through dominant alleles, haploinsufficiency, gene duplication and aneuploidy. Dominant-acting, missense alleles in all three subunits of yeast AMP-activated protein kinase confer resistance to 2-deoxyglucose. Dominant-acting, nonsense alleles in the REG1 gene, which encodes a negative regulator of AMP-activated protein kinase, confer 2-deoxyglucose resistance through haploinsufficiency. Most of the resistant strains isolated in this study achieved resistance through aneuploidy. Cells with a monosomy of chromosome 4 are resistant to 2-deoxyglucose. While this genetic strategy comes with a severe fitness cost, it has the advantage of being readily reversible when 2-deoxyglucose selection is lifted. Increased expression of the two DOG phosphatase genes on chromosome 8 confers resistance and was achieved through trisomies and tetrasomies of that chromosome. Finally, resistance was also mediated by increased expression of hexose transporters, achieved by duplication of a 117 kb region of chromosome 4 that included the HXT3, HXT6 and HXT7 genes. The frequent use of aneuploidy as a genetic strategy for drug resistance in diploid yeast and human tumors may be in part due to its potential for reversibility when selection pressure shifts.

Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis.

Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.

Changes in the expression of genes encoding type IV pili-associated proteins are seen when Clostridium perfringens is grown in liquid or on surfaces.

BACKGROUND: Clostridium perfringens is a Gram-positive anaerobic pathogen that causes multiple diseases in humans and animals. C. perfringens lack flagella but have type IV pili (TFP) and can glide on agar surfaces. When C. perfringens bacteria are placed on surfaces, they become elongated, flexible and have TFP on their surface, traits not seen in liquid-grown cells. In addition, the main pilin in C. perfringens TFP, PilA2, undergoes differential post-translational modification when grown in liquid or on plates. To understand the mechanisms underlying these phenotypes, bacteria were grown in three types of liquid media and on agar plates with the same medium to compare gene expression using RNA-Seq. RESULTS: Hundreds of genes were differentially expressed, including transcriptional regulatory protein-encoding genes and genes associated with TFP functions, which were higher on plates than in liquid. Transcript levels of TFP genes reflected the proportion of each protein predicted to reside in a TFP assembly complex. To measure differences in rates of translation, the Escherichia coli reporter gene gusA gene (encoding ß-glucuronidase) was inserted into the chromosome downstream of TFP promoters and in-frame with the first gene of the operon. ß-glucuronidase expression was then measured in cells grown in liquid or on plates. ß-glucuronidase activity was proportional to mRNA levels in liquid-grown cells, but not plate-grown cells, suggesting significant levels of post-transcriptional regulation of these TFP-associated genes occurs when cells are grown on surfaces. CONCLUSIONS: This study reveals insights into how a non-flagellated pathogenic rod-shaped bacterium senses and responds to growth on surfaces, including inducing transcriptional regulators and activating multiple post-transcriptional regulatory mechanisms associated with TFP functions.