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

Monoclonal antibodies were used to monitor the accumulation of the major capsid protein of the lactococcal small isometric bacteriophage u136 (P335 species) over the course of a one-step growth curve. A sandwich enzyme-linked immunosorbent assay was then used to distinguish two abortive phage resistance mechanisms, Hsp and Prf. Capsid protein production of u136 was almost totally inhibited by the Hsp-induced abortive mechanism, supporting previous data that this mechanism blocks phage DNA replication. Prf-induced abortive infection only partially (50%) inhibited capsid protein production, suggesting that this mechanism targets some other point, perhaps within transcription or translation processes. The results confirmed that Hsp and Prf act at different targets in the phage lytic cycle. Use of monoclonal antibodies also demonstrated that production of the major capsid protein is a nonlimiting step in the lytic cycle of lactococcal phage u136.

Differentiation of Two Abortive Mechanisms by Using Monoclonal Antibodies Directed toward Lactococcal Bacteriophage Capsid Proteins.

The role and potential of probiotic bacteria in the gut, and the communication between gut microflora and gut/host

The fifth phage resistance factor from the prototype phage-insensitive strain Lactococcus lactis subsp. lactis ME2 has been characterized and sequenced. The genetic determinant for Prf (phage resistance five) was subcloned from the conjugative plasmid pTN20, which also encodes a restriction and modification system. Typical of other abortive resistance mechanisms, Prf reduces the efficiency of plaquing to 10(-2) to 10(-3) and decreases the plaque size and burst size of the small isometric-headed phage p2 in L. lactis subsp. lactis LM0230. However, normal-size plaques occurred at a frequency of 10(-4) and contained mutant phages that were resistant to Prf, even after repeated propagation through a sensitive host. Prf does not prevent phage adsorption or promote restriction and modification activities, but 90% of Prf+ cells infected with phage p2 die. Thus, phage infections in Prf+ cells are aborted. Prf is effective in both L. lactis subsp. lactis and L. lactis subsp. cremoris strains against several small isometric-headed phages but not against prolate-headed phages. The Prf determinant was localized by Tn5 mutagenesis and subcloning. DNA sequencing identified a 1,056-nucleotide structural gene designated abiC. Prf+ expression was obtained when abiC was subcloned into the lactococcal expression vector pMG36e. abiC is distinct from two other lactococcal abortive phage resistance genes, abiA (Hsp+, from L. lactis subsp. lactis ME2) and abi416 (Abi+, from L. lactis subsp. lactis IL416). Unlike abiA, the action of abiC does not appear to affect DNA replication. Thus, abiC represents a second abortive system found in ME2 that acts at a different point of the phage lytic cycle.

Molecular characterization of a second abortive phage resistance gene present in Lactococcus lactis subsp. lactis ME2.

Bacteriophage resistance mechanisms which are derived from a bacteriophage genome are termed Per (phage-encoded resistance). When present in trans in Lactococcus lactis NCK203, Per50, the cloned origin of replication from phage phi50, interferes with phi50 replication. The per50 fragment was found to afford negligible protection to NCK203 against phi50 infection when present in a low-copy-number plasmid, pTRK325. A high-copy-number Per50 construct (pTRK323) dramatically affected phi50 infection, reducing the efficiency of plaquing (EOP) to 2.5 x 10 and the plaque size to pinhead proportions. This clone also afforded significant protection against other related small isometric phages. Per31 was cloned from phage phi31 and demonstrated to function as an origin of replication by enabling replication of per31-containing plasmids, in NCK203, on phi31 infection. A low-copy-number Per31 plasmid (pTRK360) reduced the EOP of phi31 on NCK203 to 0.3 and the plaque diameter from 1.5 to 0.5 mm. When this plasmid was cloned in high copy number, the EOP was further reduced to 7.2 x 10 but the plaques were large and contained Per31-resistant phages. Characterization of these "new" phages revealed at least two different types that were similar to phi31, except that DNA alterations were noted in the region containing the origin. This novel and powerful abortive phage resistance mechanism should prove useful when directed at specific, problematic phages.

Effect of Increasing the Copy Number of Bacteriophage Origins of Replication, in trans, on Incoming-Phage Proliferation.

Background: Lactose intolerance (LI) is a common medical problem with limited treatment options. The primary symptoms are abdominal pain, diarrhea, bloating, flatulence, and cramping. Limiting dairy foods to reduce symptoms contributes to low calcium intake and the risk for chronic disease. Adaptation of the colon bacteria to effectively metabolize lactose is a novel and potentially useful approach to improve lactose digestion and tolerance. RP-G28 is novel galacto-oligosaccharide (GOS) being investigated to improve lactose digestion and the symptoms of lactose intolerance in affected patients. Methods: A randomized, double-blind, parallel group, placebo-controlled study was conducted at 2 sites in the United States. RP-G28 or placebo was administered to 85 patients with LI for 35 days. Post-treatment, subjects reintroduced dairy into their daily diets and were followed for 30 additional days to evaluate lactose digestion as measured by hydrogen production and symptom improvements via a patient-reported symptom assessment instrument. Results: Lactose digestion and symptoms of LI trended toward improvement on RP-G28 at the end of treatment and 30 days post-treatment. A reduction in abdominal pain was also demonstrated in the study results. Fifty percent of RP-G28 subjects with abdominal pain at baseline reported no abdominal pain at the end of treatment and 30 days post treatment (p = 0.0190). RP-G28 subjects were also six times more likely to claim lactose tolerance post-treatment once dairy foods had been re-introduced into their diets (p = 0.0389). Conclusions: Efficacy trends and favorable safety/tolerability findings suggest that RP-G28 appears to be a potentially useful approach for improving lactose digestion and LI symptoms. The concurrent reduction in abdominal pain and improved overall tolerance could be a meaningful benefit to lactose intolerant individuals.

/pdf/improving-lactose-digestion-and-symptoms-of-lactose-3hv99varyk.pdf

Improving lactose digestion and symptoms of lactose intolerance with a novel galacto-oligosaccharide (RP-G28): a randomized, double-blind clinical trial.

Todd R. Klaenhammer

Papers

Differentiation of Two Abortive Mechanisms by Using Monoclonal Antibodies Directed toward Lactococcal Bacteriophage Capsid Proteins.

The role and potential of probiotic bacteria in the gut, and the communication between gut microflora and gut/host

Molecular characterization of a second abortive phage resistance gene present in Lactococcus lactis subsp. lactis ME2.

Effect of Increasing the Copy Number of Bacteriophage Origins of Replication, in trans, on Incoming-Phage Proliferation.

Improving lactose digestion and symptoms of lactose intolerance with a novel galacto-oligosaccharide (RP-G28): a randomized, double-blind clinical trial.