Peptidoglycan hydrolases as species-specific markers to differentiate Lactobacillus helveticus from Lactobacillus gallinarum and other closely related homofermentative lactobacilli.

We propose a new method that allows accurate discrimination of Lactobacillus helveticus from other closely related homofermentative lactobacilli, especially Lactobacillus gallinarum. This method is based on the amplification by PCR of two peptidoglycan hydrolytic genes, Lhv_0190 and Lhv_0191. These genes are ubiquitous and show high homology at the intra-species level. The PCR method gave two specific PCR products, of 542 and 747 bp, for 25 L. helveticus strains coming from various sources. For L. gallinarum, two amplicons were obtained, the specific 542 bp amplicon and another one with a size greater than 1,500 bp. No specific PCR products were obtained for 12 other closely related species of lactobacilli, including the L. acidophilus complex, L. delbrueckii, and L. ultunensis. The developed PCR method provided rapid, precise, and easy identification of L. helveticus. Moreover, it enabled differentiation between the two closely phylogenetically related species L. helveticus and L. gallinarum.

Structural studies of the cell wall polysaccharides from three strains of Lactobacillus helveticus with different autolytic properties: DPC4571, BROI, and LH1.

Lactobacillus helveticus is traditionally used in dairy industry as a starter or an adjunct culture for manufacture of cheese and some types of fermented milk. Its autolysis releases intracellular enzymes which is a prerequisite for optimum cheese maturation, and is known to be strain dependent. Autolysis is caused by an enzymatic hydrolysis of the cell wall peptidoglycan (PG) by endogenous peptidoglycan hydrolases (PGHs) or autolysins. Origins of differences in autolytic properties of different strains are not fully elucidated. Regulation of autolysis possibly depends on the structure of the cell wall components other than PG, particularly polysaccharides. In the present work, we screened six L. helveticus strains with different autolytic properties: DPC4571, BROI and LH1. We established, for the first time, that cell walls (CWs) of these strains contained polysaccharides, different from their CW teichoic acids. Cell wall polysaccharides of three strains were purified, and their chemical structures were established by 2D NMR spectroscopy and methylation analysis. The structures of their repeating units are presented.

The nine peptidoglycan hydrolases genes in Lactobacillus helveticus are ubiquitous and early transcribed.

Peptidoglycan hydrolases (PGHs) are bacterial enzymes that can hydrolyze the peptidoglycan in bacterial cell wall leading to autolysis. By releasing intracellular enzymes, autolysis of Lactobacillus helveticus has important applications in cheese ripening as its extent varied from strain to strain. Nine genes coding PGHs were previously annotated in the genome of the high autolytic strain L. helveticus DPC 4571. This study was conducted to evaluate the clone diversity of the nine PGHs genes within a collection of 24 L. helveticus strains, highly diverse in terms of origin, biotope and autolytic activity. Pulsed field gel electrophoresis was applied to assess the genomic diversity of the 24 strains. The presence or absence of nine PGHs genes was verified for all L. helveticus strains. Nucleotide and deduced amino acid sequence were compared for six relevant strains. Finally, gene expression was monitored by reverse transcription during growth and by zymogram for 12 strains. The nine PGHs genes are ubiquitous and transcripted early during growth. Zymograms were similar in terms of molecular size of the bands, but exhibited strain to strain variations in the number of bands revealing from 2 to 5 lytic bands per strain.