Analysis of CRISPR-Cas Loci and their Targets in Levilactobacillus brevis.

The CRISPR‒Cas system acts as a bacterial defense mechanism by conferring adaptive immunity and limiting genetic reshuffling. However, under adverse environmental hazards, bacteria can employ their CRISPR‒Cas system to exchange genes that are vital for adaptation and survival. Levilactobacillus brevis is a lactic acid bacterium with great potential for commercial purposes because it can be genetically manipulated to enhance its functionality and nutritional value. Nevertheless, the CRISPR‒Cas system might interfere with the genetic modification process. Additionally, little is known about the CRISPR‒Cas system in this industrially important microorganism. Here, we investigate the prevalence, diversity, and targets of CRISPR‒Cas systems in the genus Levilactobacillus, further focusing on complete genomes of L. brevis. Using the CRISPRCasFinder webserver, we identified 801 putative CRISPR-Cas systems in the genus Levilactobacillus. Further investigation focusing on the complete genomes of L. brevis revealed 54 putative CRISPR-Cas systems. Of these, 46 were orphan CRISPRs, and eight were CRISPR‒Cas systems. The type II-A CRISPR‒Cas system is the most common in Levilactobacillus and L. brevis complete genomes. Analysis of the spacer's target showed that the CRISPR‒Cas systems of L. brevis mainly target the enterococcal plasmids. Comparative analysis of putative CRISPR-Cas loci in Levilactobacillus brevis.

Association study of angiotensin converting enzyme gene polymorphism with elderly diabetic hypertension and lipids levels.

OBJECTIVE: To investigate the relationship between angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism and diabetic essential hypertension in elderly population. METHODS: Polymerase chain reaction (PCR) technique was used in 260 elderly normal control patients, 205 elderly hypertensive patients and 138 elderly diabetic hypertensive patients to detect the I/D polymorphism in ACE gene. RESULTS: DD genotype frequency (0.352) and D allele frequency (0.543) in elderly hypertensive patients were higher than those in the normal control patients. DD genotype (0.421) and D allele frequency (0.579) in elderly diabetic hypertensive patients were significantly higher than those in the control patients (0.133 and 0.250). The differences of DD genotype and D allele frequency between the elderly hypertensive patients and the elderly diabetic hypertensive patients were not significant (P > 0.05). CONCLUSION: ACE gene deletion is a risk factor for hypertension but is not a risk factor for diabetes in elderly population.

Characterization of class 1 integron gene cassettes among clinical bacteria isolated from one large hospital in northern China.

The class 1 integron and complex gene cassettes among different species of clinical isolates in northern China were characterized in this study. 383 clinical isolates were obtained from northern China, and class 1 integrons containing gene cassettes widely distributed among gram negative clinical isolates was observed. We find that the class 1 integron showed positive correlation with multidrug resistance phenotype of gram negative bacteria. In addition, we find that isolates belonged to one species harbored different types of gene cassette arrays, while same types of gene cassette arrays were observed in different species of isolates. The diversity of gene cassette arrays among the isolates indicated the complexity of multidrug resistance in clinical isolates in northern China.

Reciprocal regulation between SigK and differentiation programs in Streptomyces coelicolor.

Here we reported that deletion of SigK (SCO6520), a sigma factor in Streptomyces coelicolor, caused an earlier switch from vegetative mycelia to aerial mycelia and higher expression of chpE and chpH than that in the wild type. Loss of SigK also resulted in accelerated and enhanced production of antibiotics, actinorhodin, and undecylprodigiosin and increased expression of actII-orf4 and redD. These results suggested that SigK had a negative role in morphological transition and secondary metabolism. Furthermore, the sigK promoter (sigKp) activity gradually increased and sigK expression was partially dependent on SigK, but this dependence decreased during the developmental course of substrate mycelia. Meanwhile, two potentially nonspecific cleavages occurred between SigK and green fluorescent protein, and the SigK fusion proteins expressed under the constitutive promoter ermEp* sharply decreased and disappeared when aerial mycelia emerged. If expressed under sigKp, 3FLAG-SigK showed similar dynamic patterns but did not decrease as sharply as SigK expressed under ermEp*. These data suggested that the climbing expression of sigK might reduce the prompt degradation of SigK during vegetative hypha development for the proper timing of morphogenesis and that SigK vanished to remove the block for the emergence of aerial mycelia. Thus, we proposed that SigK had inhibitory roles on developmental events and that these inhibitory effects may be released by SigK degradation.

Involvement of SigT and RstA in the differentiation of Streptomyces coelicolor.

SigT is an ECF sigma factor in Streptomyces coelicolor. sigT and its putative anti-sigma factor gene rstA are located in one putative operon, and SigT could physically interact with RstA. Deletion of sigT or rstA caused accelerated morphological development and enhanced production of antibiotics, concomitant with over-expression of chpE, chpH, actII-orf4 and redD. Furthermore, SigT was undetectable after loss of rstA. These data suggested that SigT has a negative role on differentiation and that RstA negatively regulates the SigT activity through a putative antagonistic mechanism and at the post-transcriptional level.

New approach to achieve high-level secretory expression of heterologous proteins by using Tat signal peptide.

The twin-arginine translocation (Tat) pathway is an attractive route for secretory production of heterologous proteins in E. coli. In this study, we investigated the potential use of Tat signal peptide from S. coelicolor to improve secretory expression. The results showed that Tat signal peptide (ssDagA) could effectively secrete active Green fluorescent protein (GFP) to periplasm. When the rare codons of signal sequence were optimized, the expression and secretion yield of GFP improved by about 2-3 folds as detected qualitatively by western blotting and fluorescent analysis. The increase of translation rate could be explained by the unstability of mRNA secondary structure. In summary, our strategy could provide a new approach for high-level secretory expression of heterologous proteins in E. coli.

FtsY affects sporulation and antibiotic production by whiH in Streptomyces coelicolor.

FtsY, the Signal Recognition Particle (SRP) receptor in bacteria, is known to facilitate the cotranslational protein targeting by recruiting SRP-protein complex to secYEG. We show in this work that deletion of the ftsY gene in Streptomyces coelicolor would also lead to complete blockage of sporulation process and reduced production of antibiotic actinorhodin. These defects cannot be complemented by only the NG domain of FtsY, while full-length ftsY was able to restore spore generation and increase production of actinorhodin in ftsY-disrupted strains. Further transcriptional analysis on sporulation controlling genes, i.e., whiG, whiB, whiH, and prox, indicated that the regulation of sporulation by ftsY is likely to take effect through whiH.

Roles of larger conductance mechanosensitive channels (MscL) in sporulation and Act secretion in Streptomyces coelicolor.

Larger conductance mechanosensitive channels (MscL) is a conserved channel existing in eukarya, bacteria and archaea. The gene SCO3190 in Streptomyces coelicolor encodes an MscL homolog of Escherichia coli. To elucidate the physiological roles of MscL in S. coelicolor (Sc-MscL), deletion mutant was constructed. The Sc-MscL deletion brought sporulation ahead, and had no significant influence to antibiotic synthesis in S. coelicolor. When Sc-MscL was overexpressed in S. coelicolor, the colony of S. coelicolor was small and sleek with abundant blue pigment corresponding to actinorhodin (Act). Furthermore, abundant blue pigment displayed on hypoosmotic LB plates comparing to hyperosmotic R5 plates. These results suggested that Sc-MscL was involved into Act secretion and sporulation relating to osmotic stress response in S. coelicolor. Moreover, Sc-MscL showed possible functional pentamer by cross-linking in vitro. It is first report about biochemical and functional characterization of MscL in S. coelicolor.

[Preparation of monodispersed agglomerated pellicular cation exchange resins and its chromatographic properties].

A new and simple method for the preparation of latex-coated pellicular cation exchanger is introduced. Chromatographic stationary phase of monodispersed agglomerated pellicular cation was prepared for the first time. The monodispersed polyvinylbenzene-divinylbenzene (PSTDVB) particles (10 microns) as substrate beads were functionalized with a tertiary amine on the surface at first and then attached with a layer of sulfonated latex particles (0.2 micron) to give the desired packing. Its chromatographic properties were evaluated by using alkali metals (Li+, Na+, NH4+, K+) and alkaline earth metals (Mg2+, Ca2+). The results showed that the packing is of good resolution, low detection limit, and excellent repeatability for common inorganic cations.