Motivation: In 2001 and 2002, we published two papers (Bioinformatics, 17, 282--283, Bioinformatics, 18, 77--82) describing an ultrafast protein sequence clustering program called cd-hit. This program can efficiently cluster a huge protein database with millions of sequences. However, the applications of the underlying algorithm are not limited to only protein sequences clustering, here we present several new programs using the same algorithm including cd-hit-2d, cd-hit-est and cd-hit-est-2d. Cd-hit-2d compares two protein datasets and reports similar matches between them; cd-hit-est clusters a DNA/RNA sequence database and cd-hit-est-2d compares two nucleotide datasets. All these programs can handle huge datasets with millions of sequences and can be hundreds of times faster than methods based on the popular sequence comparison and database search tools, such as BLAST.

Availability: http://cd-hit.org

Contact: [email protected]

Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences

Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.

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CRISPR provides acquired resistance against viruses in prokaryotes

Evolution of Protein Molecules

Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from each other using molecular methods including DNA-DNA hybridization with oligonucleotide probes and DNA sequencing. A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA. New tetracycline derivatives are being examined, although their role in treatment is not clear. Changing the use of tetracyclines in human and animal health as well as in food production is needed if we are to continue to use this class of broad-spectrum antimicrobials through the present century.

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

Obesity and type 2 diabetes are characterized by altered gut microbiota, inflammation, and gut barrier disruption. Microbial composition and the mechanisms of interaction with the host that affect gut barrier function during obesity and type 2 diabetes have not been elucidated. We recently isolated Akkermansia muciniphila, which is a mucin-degrading bacterium that resides in the mucus layer. The presence of this bacterium inversely correlates with body weight in rodents and humans. However, the precise physiological roles played by this bacterium during obesity and metabolic disorders are unknown. This study demonstrated that the abundance of A. muciniphila decreased in obese and type 2 diabetic mice. We also observed that prebiotic feeding normalized A. muciniphila abundance, which correlated with an improved metabolic profile. In addition, we demonstrated that A. muciniphila treatment reversed high-fat diet-induced metabolic disorders, including fat-mass gain, metabolic endotoxemia, adipose tissue inflammation, and insulin resistance. A. muciniphila administration increased the intestinal levels of endocannabinoids that control inflammation, the gut barrier, and gut peptide secretion. Finally, we demonstrated that all these effects required viable A. muciniphila because treatment with heat-killed cells did not improve the metabolic profile or the mucus layer thickness. In summary, this study provides substantial insight into the intricate mechanisms of bacterial (i.e., A. muciniphila) regulation of the cross-talk between the host and gut microbiota. These results also provide a rationale for the development of a treatment that uses this human mucus colonizer for the prevention or treatment of obesity and its associated metabolic disorders.

https://www.pnas.org/content/pnas/110/22/9066.full.pdf

Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity

Recombinant bacteria containing nucleotide sequences encoding Streptococcus thermophilus phage helicase and primase genes expressed in the antisense orientation and methods of making and using the same are disclosed. Replication of bacteriophage infecting this bacterial cell, can be inhibited or decreased by constitutively expressing, the antisense oligonucleotide encoding for S. thermophilus phage helicase or primase.

Antisense rna expression strategies effective against streptococcus thermophilus bacteriophage

Advances in the microbiology and biochemistry of cheese and fermented milk: edited by F. Lyndon Davies and Barry A. Law, Elsevier Applied Science Publishers, 1984. £28.00 (viii + 260 pages) ISBN 0 85334 287 3

Effect of Green Tea and EGCG on the Growth Rate and Cell Density of the Probiotic Bacteria Lactobacillus acidophilus and Lactobacillus gasseri

We have recently developed a novel bacteriophage-protection system for Lactococcus lactis, based on a two-component genetic ‘trap’. An inducible promoter from a lytic bacteriophage is used to activate a lethal gene after infection, killing the host cell and halting phage proliferation. To expand the potential use of this novel defense strategy, promoters specific to any particular phage of interest must be available for fusion to a universal death gene. A method to localize regulated promoters within the context of the total phage genome was evaluated. The ‘capping’ activity of the vaccinia virus guanylyltransferase was exploited to label newly synthesized mRNA extracted from infected cells at sequential time points over the course of a phage infection. The labeled mRNAs were then used as probes in Southern hybridization reactions to identify restriction fragments in the phage genome where new transcripts were initiated during progression of the phage lytic cycle. This method has been used successfully in our laboratory to map the general location of a number of inducible promoters on the genomes of bacteriophages attacking lactic acid bacteria. Once identified and cloned, small fragments encoding inducible promoters can be partially sequenced and primer extension reactions carried out on phage RNA, isolated over the course of an infection, to pinpoint the precise location of the promoter. In this study, we illustrate the use of the capping method to map the phage-inducible promoter on the genome of the lactococcal bacteriophage skl. This approach provides a rapid and efficient means to identify promoter regions on the genomes of relatively uncharacterized phages. These promoters can then be used in a variety of applications, including phage-triggered defenses and inducible gene expression systems.

A method for mapping phage-inducible promoters for use in bacteriophage-triggered defense systems

[] Glycogen, a soluble multi-branched glucose homopolysaccharide, is composed of chains of -1,4-linked glucose residues interconnected by -1,6-linked branches. The classical biosynthetic pathway involves phosphoglucomutase (Pgm), glucose-1-phosphate adenylyltransferase (GlgC or GlgCD), glycogen synthase (GlgA) and branching enzyme (GlgB). Phosphoglucomutase converts glucose-6-phosphate into glucose-1-phosphate, which serves as a substrate for ADP-glucose synthesis catalyzed by GlgC or GlgCD. Then, GlgA catalyzes the transfer of glucosyl units from ADP-glucose to the elongating chain of linear -1,4-glucan. GlgB subsequently cleaves off portions of the glucan and links it to internal glucose molecules in existing chains via -1,6 glycosidic bonds to form the glycogen structure. Glycogen breakdown is mediated by glycogen phosphorylase (GlgP) and debranching enzyme (GlgX), which catalyze the sequential phosphorolysis of -1,4-glucosyl linkages in the glucan chain from the non-reducing ends and debranching of the limit dextrins generated by GlgP, respectively. An increasing number of studies have revealed the involvement of glycogen metabolism in a multitude of physiological functions in some prokaryotes beyond the function of synthesizing energy storage compounds. Lactobacillus acidophilus NCFM was the first probiotic lactic acid bacterium demonstrated to possess a functional glycogen biosynthesis pathway that is involved in its growth, bile tolerance and complex carbohydrate metabolism (Goh and Klaenhammer, 2013). The following qualitative (for detection of intracellular glycogen) and quantitative (for measurement of intracellular glycogen content) intracellular glycogen assay protocols for Lactobacillus acidophilus (L. acidophilus) were modified from previous works (Govons et al., 1969; Law et al., 1995; Parrou and Francois, 1997) and should be applicable to other lactic acid bacteria as well as most microorganisms.

/pdf/intracellular-glycogen-assays-184moocnht.pdf

Todd R. Klaenhammer

Papers

Antisense rna expression strategies effective against streptococcus thermophilus bacteriophage

Advances in the microbiology and biochemistry of cheese and fermented milk: edited by F. Lyndon Davies and Barry A. Law, Elsevier Applied Science Publishers, 1984. £28.00 (viii + 260 pages) ISBN 0 85334 287 3

Effect of Green Tea and EGCG on the Growth Rate and Cell Density of the Probiotic Bacteria Lactobacillus acidophilus and Lactobacillus gasseri

A method for mapping phage-inducible promoters for use in bacteriophage-triggered defense systems

Intracellular Glycogen Assays