Ribosome recycling factor (RRF), a product of the frr gene, is responsible for the dissociation of ribosomes from messenger RNA after the termination of translation. In order to overexpress frr gene in the toyocamycin (TM) producer Streptomyces diastatochromogenes 1628, we cloned and placed the gene under the control of the constitutive promoter PermE*. The resulting plasmid pIB139-frr was integrated into the chromosome of S. diastatochromogenes 1628 by conducting intergeneric conjugation. The strain S. diastatochromogenes 1628 containing pIB139-frr (1628-FRR) showed a 33.3 % increase in cell growth and a 46 % increase in TM production compared to wild-type strain 1628 when cultivated in a 7 l fermentor. In addition, it was possible to shorten the fermentation time from 84 to 72 h. Furthermore, by conducting reverse transcription polymerase chain reaction (RT-PCR) analysis, we discovered that the transcriptional levels of regulatory gene adpA-sd, toyF, and toyG involved in TM biosynthesis were enhanced in S. diastatochromogenes 1628-FRR compared to S. diastatochromogenes 1628. In addition, by using a fluorescent intensity reporter system, which is based on the green fluorescent protein (GFP), and by using Western blot analysis, we revealed that overexpression of frr also strongly promoted protein biosynthesis in late growth phase. These findings confirmed that by increasing copy number of frr gene, it is a useful approach to improve antibiotic production.

Overexpression of ribosome recycling factor is responsible for improvement of nucleotide antibiotic-toyocamycin in Streptomyces diastatochromogenes 1628.

In this study, four galactose-positive (Gal+) Streptococcus thermophilus strains viz. AJM, JM1, KM3 and AUKD8 and one galactose-negative (Gal−) S. thermophilus NCDC 218 were used to characterize the organization of Leloir pathway genes using long-range PCR, and expression of these genes were studied using real-time PCR, in presence of different sugars. Long-range PCR results showed that both Gal+ and Gal− isolates, the gal–lac gene order (galRKTEM–lacSZ), are conserved including the size of individual genes. The promoter sequence of the three Gal+ isolates (AJM, JM1 and KM3) possessed single base pair deletion at −28 region of galR and C to T substitution at −9 box galK region. In contrast, Gal+ AUKD8 had A to T substitution at preceding −25 region of galR. The expression of galK and galM grown in the presence of galactose was significantly higher in case of AJM (30- and 7.6-fold, respectively), followed by KM3 and JM1. In addition, galR, galT and galE showed higher expression in galactose, than in lactose and glucose medium. This study gives a preliminary idea on Leloir pathway gene expression in wild Gal+
 S. thermophilus, and further studies may throw more light on the role of gal–lac operon in galactose metabolism.

Assessment of expression of Leloir pathway genes in wild-type galactose-fermenting Streptococcus thermophilus by real-time PCR

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms.

https://aem.asm.org/content/aem/81/20/7244.full.pdf

Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method

Single-cell methods are a powerful application in microbial research to study the molecular mechanism underlying phenotypic heterogeneity and cell-to-cell variability. Here, we describe the optimization and application of single-cell time-lapse fluorescence microscopy for the food spoilage bacterium Bacillus cereus specifically. This technique is useful to study cellular development and adaptation, gene expression, protein localization, protein mobility, and cell-to-cell communication over time at the single-cell level. By adjusting existing protocols, we have enabled the visualization of growth and development of single B. cereus cells within a microcolony over time. Additionally, several different fluorescent reporter proteins were tested in order to select the most suitable green fluorescent protein (GFP) and red fluorescent protein (RFP) candidates for visualization of growth stage- and cell compartment-specific gene expression in B. cereus. With a case study concerning cotD expression during sporulation, we demonstrate the applicability of time-lapse fluorescence microscopy. It enables the assessment of gene expression levels, dynamics, and heterogeneity at the single-cell level. We show that cotD is not heterogeneously expressed among cells of a subpopulation. Furthermore, we discourage using plasmid-based reporter fusions for such studies, due to an introduced heterogeneity through copy number differences. This stresses the importance of using single-copy integrated reporter fusions for single-cell studies.

/pdf/live-cell-imaging-tool-optimization-to-study-gene-expression-4qketimpvz.pdf

Live-cell imaging tool optimization to study gene expression levels and dynamics in single cells of Bacillus cereus.

Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Forster Resonance Energy Transfer (FRET). Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM). For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV)-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.

/pdf/single-cell-fret-analysis-for-the-identification-of-optimal-2f1301fem8.pdf

Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.

Lactococcus lactis MG1363 Appearance of Lactose-Utilizing Cluster Accounts for the cel of the Silent A Specific Mutation in the Promoter Region

The present invention relates to the field of microbiology, specifically the field of production of a dairy food product using fermentation and the field of gene transfer by conjugation.

A method for the production of a dairy food product and a method for gene transfer by conjugation

Benchmarking various GFP variants in Bacillus subtilis

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Lactococcus lactis MG1363 Appearance of Lactose-Utilizing Cluster Accounts for the cel of the Silent A Specific Mutation in the Promoter Region

A method for the production of a dairy food product and a method for gene transfer by conjugation

Benchmarking various GFP variants in Bacillus subtilis