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.

/pdf/crispr-provides-acquired-resistance-against-viruses-in-255k92gy6w.pdf

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

Genes and gene expression in Lactococcus bacteriophages

A rapid and simple technique was developed for conjugation between group N and group D streptococci by using cells entrapped within calcium alginate gel beads. With this method, the frequencies of transfer of lactose metabolism from Streptococcus lactis ME2 to S. lactis LM2302 were comparable to those achieved with agar surface matings. Conjugal transfer of the chloramphenicol and erythromycin resistance plasmid pVA797::Tn917 from S. faecalis V1229 to S. faecalis V1102 in alginate beads occurred at frequencies comparable to those achieved with filter matings. The results demonstrated efficient conjugal transfer of plasmid DNA among alginate-immobilized streptococcal cells and suggested that this method could be used as an alternative to conventional solid-surface and filter matings with these organisms.

Conjugal transfer of plasmid DNA between streptococci immobilized in calcium alginate gel beads.

(Lac') transconjugants generated frommatings between S.lactis Ni,anME2 variant, andaplasmid-free recipient, S.lactis LM2301.Theplaquing efficiencies ofprolate- andsmall isometric-headed phages werereduced ontransconjugants containing either pTN20(R+/M+Tra+) or100-kb plasmids encoding Lac', R+/M+,andTra+.Lac'transconjugants whichharbored pTR1040(Lac') and pTN20(R+/M+) werephenotypically R-/M-andtransferred Lac'atlowfrequency insubsequent matings to giverise to100-kb R+/M+plasmids. R+/M+activities andhigh-frequency conjugal transfer ability were detected inLac'transconjugants thatcontained pTR1041(Lac') andpTN20(R+/M+). No100-kb R+/M+ plasmids wererecovered after these matings, suggesting thatpTR1041wasmobilized bypTN20through a process thatresembled plasmid donation. pTR1041wasidentical topTR1040 butcontained anadditional 3.3-kb DNA fragment. Thesedatasuggested thatphenotypic expression ofR+/M+andTra+isaffected by coresident Lac'plasmids. Restriction enzymeanalysis andhybridization reactions demonstrated thatthe 100-kb R+/M+plasmid wasformed byacointegration event between pTR1040(Lac+) andpTN20(R+/M+ Tra+)during conjugal transfer viaa conductive-type process. Thisisthefirst report thatdefines selftransmissible restriction andmodification plasmids inthelactic streptococci.

Restriction andModification Activities fromStreptococcus lactis ME2AreEncoded byaSelf-Transmissible Plasmid, pTN20,ThatFormsCointegrates during Mobilization ofLactose-Fermenting Abilityt

C.LlaI is a bifunctional regulatory protein of the llaI restriction modification operon from Lactococcus lactis.

Foods are teeming with microorganisms that may be innocuous, pathogenic threats, spoilage agents, or beneficial microorganisms driving fermentations or acting as biocontrol agents. This chapter outlines the basic concepts underlying genomics, proteomics, and associated technologies. With the number of completely sequenced bacterial genomes increasing rapidly, one powerful approach to defining unique or conserved gene content and understanding how Lactobacillus plantarum microorganisms evolved is comparative genomics, via an in silico analysis. The discipline of functional genomics deals with defining the roles of genes in their appropriate organisms. This chapter utilizes comparative and functional genomics and proteomics to demonstrate the role of SpaC in mucin binding and potentially its importance to the retention of some lactobacilli in the gastrointestinal tract. The principles behind DNA microarray technology make it very applicable to many different uses that include comparative genomics and global gene expression analysis. While the details of the Gad system were elucidated primarily by experiments in Escherichia coli, genomics and bioinformatics have enabled researchers to identify and study the effects of these genes in other organisms. The contribution of genome sequencing and functional genomics has greatly facilitated our understanding of the pathogenicity of Listeria monocytogenes. Differences between the pathogenic and nonpathogenic Listeria species appear most strongly in the secretory proteome.

Todd R. Klaenhammer

Papers

Genes and gene expression in Lactococcus bacteriophages

Conjugal transfer of plasmid DNA between streptococci immobilized in calcium alginate gel beads.

Restriction andModification Activities fromStreptococcus lactis ME2AreEncoded byaSelf-Transmissible Plasmid, pTN20,ThatFormsCointegrates during Mobilization ofLactose-Fermenting Abilityt

C.LlaI is a bifunctional regulatory protein of the llaI restriction modification operon from Lactococcus lactis.

Genomics and Proteomics of Foodborne Microorganisms