Pathogen-specific antibiotics kill the offending species without inviting the patient's flora to help develop a resistance mechanism. The current scarcity of pathogen-specific antibiotics reflects the rarity of essential genes that are also not widely represented in and conserved among species. The FemX enzyme that initiates the synthesis of the interchain peptide of the peptidoglycan in a subset of bacterial species was purified from Lactobacillus viridescens. Subsequently, the encoding femX gene was cloned and sequenced using reverse genetics. The femX gene is a member of the femAB family, a large family of genes previously implicated in interchain peptide synthesis but with unknown specific functions. Mutagenesis of the femX gene identified the members of the extended FemABX family as novel nonribosomal peptidyltransferases. Determinants of FemX complex substrate recognition and a strong stimulator of FemX activity were also identified. The FemABX family members are ideal candidates for pathogen-specific antibiotic development.

Weissella viridescens FemX (FemX(Wv)) belongs to the Fem family of nonribosomal peptidyl transferases that use aminoacyl-tRNA as the amino acid donor to synthesize the peptide cross-bridge found in the peptidoglycan of many species of pathogenic gram-positive bacteria. We have recently solved the crystal structure of FemX(Wv) in complex with the peptidoglycan precursor UDP-MurNAc-pentapeptide and report here the site-directed mutagenesis of nine residues located in the binding cavity for this substrate. Two substitutions, Lys36Met and Arg211Met, depressed FemX(Wv) transferase activity below detectable levels without affecting protein folding. Analogues of UDP-MurNAc-pentapeptide lacking the phosphate groups or the C-terminal D-alanyl residues were not substrates of the enzyme. These results indicate that Lys36 and Arg211 participate in a complex hydrogen bond network that connects the C-terminal D-Ala residues to the phosphate groups of UDP-MurNAc-pentapeptide and constrains the substrate in a conformation that is essential for transferase activity.

Members of the FemABX protein family are novel therapeutic targets, as they are involved in the synthesis of the bacterial cell wall. They catalyze the addition of amino acid(s) on the peptidoglycan precursor using aminoacylated tRNA as a substrate. We report here the high-resolution structure of Weissella viridescens L-alanine transferase FemX and its complex with the UDP-MurNAc-pentapeptide. This is the first structure example of a FemABX family member that does not possess a coiled-coil domain. FemX consists of two structurally equivalent domains, separated by a cleft containing the binding site of the UDP-MurNAc-pentapeptide and a long channel that traverses one of the two domains. Our structural studies bring new insights into the evolution of the FemABX and the related GNAT superfamilies, shed light on the recognition site of the aminoacylated tRNA in Fem proteins, and allowed manual docking of the acceptor end of the alanyl-tRNAAla.

Two lactic acid bacteria, strains 257(T) and 252, were isolated from traditional heap fermentations of Ghanaian cocoa beans. 16S rRNA gene sequence analysis of these strains allocated them to the genus Weissella, showing 99.5 % 16S rRNA gene sequence similarity towards Weissella ghanensis LMG 24286(T). Whole-cell protein electrophoresis, fluorescent amplified fragment length polymorphism fingerprinting of whole genomes and biochemical tests confirmed their unique taxonomic position. DNA-DNA hybridization experiments towards their nearest phylogenetic neighbour demonstrated that the two strains represent a novel species, for which we propose the name Weissella fabaria sp. nov., with strain 257(T) (=LMG 24289(T) =DSM 21416(T)) as the type strain. Additional sequence analysis using pheS gene sequences proved useful for identification of all Weissella-Leuconostoc-Oenococcus species and for the recognition of the novel species.

A taxonomic study was made of the genus Leuconostoc. The species in the genus were divided into three subclusters by phylogenetic analysis based on the 16S rRNA gene sequences. The three subclusters were the Leuconostoc mesenteroides subcluster (comprising L. carnosum, L. citreum, L. gasicomitatum, L. gelidum, L. inhae, L. kimchii, L. lactis, L. mesenteroides and L. pseudomesenteroides), the L. fructosum subcluster (L. durionis, L. ficulneum, L. fructosum and L. pseudoficulneum) and the L. fallax subcluster (L. fallax). Phylogenetic trees based on the sequences of the 16S-23S rRNA gene intergenic spacer region, the rpoC gene or the recA gene indicated a good correlation with the phylogenetic tree based on 16S rRNA gene sequences. The species in the L. fructosum subcluster were morphologically distinguishable from the species in the L. mesenteroides subcluster and L. fallax as species in the L. fructosum subcluster had rod-shaped cells. In addition, the four species in the L. fructosum subcluster needed an electron acceptor for the dissimilation of d-glucose and produced acetic acid from d-glucose rather than ethanol. On the basis of evidence presented in this study, it is proposed that the four species in the L. fructosum subcluster, Leuconostoc durionis, Leuconostoc ficulneum, Leuconostoc fructosum and Leuconostoc pseudoficulneum, should be transferred to a novel genus, Fructobacillus gen. nov., as Fructobacillus durionis comb. nov. (type strain D-24(T)=LMG 22556(T)=CCUG 49949(T)), Fructobacillus ficulneus comb. nov. (type strain FS-1(T)=DSM 13613(T)=JCM 12225(T)), Fructobacillus fructosus comb. nov. (type strain IFO 3516(T)=DSM 20349(T)=JCM 1119(T)=NRIC 1058(T)) and Fructobacillus pseudoficulneus comb. nov. (type strain LC-51(T)=DSM 15468(T)=CECT 5759(T)). The type species of the genus Fructobacillus is Fructobacillus fructosus gen. nov., comb. nov.. No significant physiological and biochemical differences were found between the species in the L. mesenteroides subcluster and L. fallax in the present study and thus L. fallax remains as a member of the genus Leuconostoc.

Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.

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