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

The only major structural protein (35 kDa) of the lactococcal small isometric-headed bacteriophage ul36, a member of the P335 species, was isolated from a preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monoclonal antibodies (MAbs) were raised against the denatured 35-kDa protein. Six MAbs were selected and characterized. Western blots (immunoblots) showed that all MAbs recognized the 35 kDa but also a 45 kDa that is in lower concentration in the phage structure. Binding inhibition assays identified five families of MAbs that recognized nonoverlapping epitopes of the 35- and 45-kDa proteins. Immunoelectron microscopy showed that these two proteins are localized within the phage head, therefore indicating that the 35 kDa is a major capsid protein of ul36 and that the 45 kDa is a minor capsid protein. With two MAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for direct detection of lactococcal phages in whey and milk samples. Whey and milk components, however, interfered with the conduct of the assay. Partial denaturation of milk samples by heat treatment in the presence of SDS and β-mercaptoethanol removed the masking effect and increased the sensitivity of the assay by 100-fold. With the method used here, 107 PFU/ml were detected by the ELISA within 2 h without any steps to enrich or isolate bacteriophages.

Production of Monoclonal Antibodies against the Major Capsid Protein of the Lactococcus Bacteriophage ul36 and Development of an Enzyme-Linked Immunosorbent Assay for Direct Phage Detection in Whey and Milk.

Lactobacillus acidophilus NCFM is a probiotic microbe with the ability to survive passage to the -gastrointestinal tract, interact intimately with the host and induce immune responses. The genome of NCFM has been determined and the bacterium is genetically accessible. Therefore, L. acidophilus has excellent potential for use as a vaccine delivery vehicle to express antigens at mucosal surfaces. Plasmids, commonly used to carry antigen encoding genes, are inherently unstable and require constant selection by antibiotics, which can be problematic for in vivo studies and clinical trials. Chromosomal expression of gene cassettes encoding antigens offers enhanced genetic stability by eliminating requirements for marker selection. This work illustrates the integration and inducible expression of the reporter gene gusA3, -encoding a β-glucuronidase (GusA3), in the L. acidophilus chromosome. A previously described upp-counterselectable gene replacement system was used to direct insertion of the gusA3 gene into an intergenic chromosomal location downstream of lacZ (LBA1462), encoding a β-galactosidase. The transcriptional activity of integrated gusA3 was evaluated by GUS activity assays using 4-methyl-umbelliferyl-β-D: -glucuronide (MUG) and was determined to be one to two orders of magnitude higher than the GusA3-negative parent, NCK1909. The successful chromosomal integration and expression of GusA3 demonstrate the potential of this method for higher levels of inducible gene expression in L. acidophilus.

Integrative Food Grade Expression System for Lactic Acid Bacteria

Among the members of lactic acid bacteria, the lactobacilli represent the most diverse genus. Individual species are ubiquitous in the environment while still occupying a multitude of specialized ecological niches. These include fermenting vegetables, meat, cereals, dairy products, and the intestinal tract of man and animals. Their metabolic capabilities and physiological characteristics are equally diverse and proper classification of many strains has occurred only recently following the development of more sophisticated biochemical and genomic classification systems. These bacteria derive their energy via either homo- or heterofermentative catabolism of carbohydrates in nutritionally complex environments. As the major end product of their metabolism, organic acids function directly as antagonists (Kashet, 1987) and lower the pH of the environment. Since lactobacilli are often more acid-tolerant than other competing bacteria, including other lactic acid bacteria, acidification of the environment promotes their ability to compete within and ultimately dominate fermenting ecosystems. In addition, the lactobacilli produce a variety of chemical and proteinaceous antimicrobials. These include hydrogen peroxide, various illcharacterized compounds (Vincent et al., 1959; Hamdan & Mikolajcik, 1974; Silva et al., 1987), antimicrobials (Talarico & Dobrogosz, 1989), and most notably, bacteriocins (Klaenhammer, 1988, 1990).

Molecular Properties of Lactobacillus Bacteriocins

Stress Adaptations of Lactic Acid Bacteria

Production of cheese and cultured dairy products relies largely on the fermentation of milk by the lactic acid bacteria. Of this group Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus serve as thermophilic starters in the processing of yogurt and of Swiss, Emmental, and Italian cheeses. Many lactobacilli are also natural inhabitants of the intestinal tract of mammals. The development of new, natural, and safe antimicrobial agents and preservative systems, as well as the construction of ‘food-grade’ cloning and expression vectors using bacteriocin determinants have recently been the focus of many research efforts.

Todd R. Klaenhammer

Papers

Production of Monoclonal Antibodies against the Major Capsid Protein of the Lactococcus Bacteriophage ul36 and Development of an Enzyme-Linked Immunosorbent Assay for Direct Phage Detection in Whey and Milk.

Integrative Food Grade Expression System for Lactic Acid Bacteria

Molecular Properties of Lactobacillus Bacteriocins

Stress Adaptations of Lactic Acid Bacteria

Helveticin J, A Large Heat-Labile Bacteriocin from Lactobacillus Helveticus