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Bacteriophage-encoded lytic enzymes control growth of contaminating Lactobacillus found in fuel ethanol fermentations.

ABSTRACT: BACKGROUND:Reduced yields of ethanol due to bacterial contamination in fermentation cultures weaken the economics of biofuel production. Lactic acid bacteria are considered the most problematic, and surveys of commercial fuel ethanol facilities have found that species of Lactobacillus are predominant. Bacteriophage lytic enzymes are peptidoglycan hydrolases that can degrade the Gram positive cell wall when exposed externally and provide a novel source of antimicrobials that are highly refractory to resistance development. RESULTS:The streptococcal phage LambdaSa2 (?Sa2) endolysin demonstrated strong lytic activity towards 17 of 22 strains of lactobacilli, staphylococci or streptococci and maintained an optimal specific activity at pH 5.5 and in the presence of ? 5% ethanol (fermentation conditions) toward L. fermentum. Lactobacillus bacteriophage endolysins LysA, LysA2 and LysgaY showed exolytic activity towards 60% of the lactobacilli tested including four L. fermentum isolates from fuel ethanol fermentations. In turbidity reduction assays LysA was able to reduce optical density >75% for 50% of the sensitive strains and >50% for the remaining strains. LysA2 and LysgaY were only able to decrease cellular turbidity by <50%. Optimal specific activities were achieved for LysA, LysA2, and LysgaY at pH 5.5. The presence of ethanol (?5%) did not reduce the lytic activity. Lysins were able to reduce both L. fermentum (BR0315-1) (?Sa2 endolysin) and L. reuteri (B-14171) (LysA) contaminants in mock fermentations of corn fiber hydrolysates. CONCLUSION:Bacteriophage lytic enzymes are strong candidates for application as antimicrobials to control lactic acid bacterial contamination in fuel ethanol fermentations.

SUBMITTER: Roach DR

PROVIDER: S-EPMC3646710 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Bacteriophage-encoded lytic enzymes control growth of contaminating Lactobacillus found in fuel ethanol fermentations.

Roach Dwayne R DR Khatibi Piyum A PA Bischoff Kenneth M KM Hughes Stephen R SR Donovan David M DM

Biotechnology for biofuels 20130207 1

<h4>Background</h4>Reduced yields of ethanol due to bacterial contamination in fermentation cultures weaken the economics of biofuel production. Lactic acid bacteria are considered the most problematic, and surveys of commercial fuel ethanol facilities have found that species of Lactobacillus are predominant. Bacteriophage lytic enzymes are peptidoglycan hydrolases that can degrade the Gram positive cell wall when exposed externally and provide a novel source of antimicrobials that are highly re ...[more]

PMID: 23390890

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Project description:BACKGROUND:Contamination of corn mash by lactic acid bacteria (LAB) reduces the efficiency of the ethanol fermentation process. The industry relies heavily on antibiotics for contamination control and there is a need to develop alternative methods. The goals of this study were to determine the diversity and abundance of bacteria contaminating commercial ethanol fermentations, and to evaluate the potential of anti-LAB bacteriophages in controlling production losses. RESULTS:Bacterial populations in 27 corn mash samples collected from nine different commercial plants were determined by pyrosequencing of 16S rRNA amplicons. The results showed that the most abundant bacteria (>50 % of total population) in 24 of the 27 samples included LAB genera such as Lactobacillus, Streptococcus, Lactococcus, Weissella, Enterococcus, and Pediococcus. Lactobacillus was identified as the most prevalent genus at all fermentation stages in all plants, accounting for between 2.3 and 93.7 % of each population and constituting the major genus (>50 %) in nine samples from five plants and the most abundant genus in five other samples. Lactobacillus species, including L. delbrueckii, L. fermentum, L. mucosae, and L. reuteri were the most well-represented species. Two bacteriophages that target L. fermentum strains from ethanol plants, vB_LfeS_EcoSau and vB_LfeM_EcoInf (EcoSau and EcoInf), were isolated and characterized as a siphophage and a myophage, respectively. Analysis of the 31,703 bp genome of EcoSau revealed its similarity to the P335-like phage group, and the 106,701 bp genome of phage EcoInf was determined to be a novel phage type despite its distant relationship to the SPO1-like phages. Addition of phages EcoSau and EcoInf to L. fermentum-contaminated corn mash fermentation models restored the yields of ethanol and reduced levels of residual glucose, lactic acid, and acetic acid to that comparable to the infection-free control. CONCLUSIONS:This study provides detailed insight into the microbiota contaminating commercial ethanol fermentations, and highlights the abundance of LAB, especially L. delbrueckii, L. fermentum, L. mucosae, and L. reuteri, in the process. This study suggests that phages with broad coverage of major LAB species can be applied directly to corn mash for antibiotic-free control of contamination in the ethanol fermentation industry.

Project description:The bacteriophage exclusion (BREX) system is a novel prokaryotic defense system against bacteriophages. To our knowledge, no study has systematically characterized the function of the BREX system in lactic acid bacteria. Lactobacillus casei Zhang is a probiotic bacterium originating from koumiss. By using single-molecule real-time sequencing, we previously identified N6-methyladenine (m6A) signatures in the genome of L. casei Zhang and a putative methyltransferase (MTase), namely, pglX This work further analyzed the genomic locus near the pglX gene and identified it as a component of the BREX system. To decipher the biological role of pglX, an L. casei Zhang pglX mutant (?pglX) was constructed. Interestingly, m6A methylation of the 5'-ACRCAG-3' motif was eliminated in the ?pglX mutant. The wild-type and mutant strains exhibited no significant difference in morphology or growth performance in de Man-Rogosa-Sharpe (MRS) medium. A significantly higher plasmid acquisition capacity was observed for the ?pglX mutant than for the wild type if the transformed plasmids contained pglX recognition sites (i.e., 5'-ACRCAG-3'). In contrast, no significant difference was observed in plasmid transformation efficiency between the two strains when plasmids lacking pglX recognition sites were tested. Moreover, the ?pglX mutant had a lower capacity to retain the plasmids than the wild type, suggesting a decrease in genetic stability. Since the Rebase database predicted that the L. casei PglX protein was bifunctional, as both an MTase and a restriction endonuclease, the PglX protein was heterologously expressed and purified but failed to show restriction endonuclease activity. Taken together, the results show that the L. casei Zhang pglX gene is a functional adenine MTase that belongs to the BREX system.IMPORTANCE Lactobacillus casei Zhang is a probiotic that confers beneficial effects on the host, and it is thus increasingly used in the dairy industry. The possession of an effective bacterial immune system that can defend against invasion of phages and exogenous DNA is a desirable feature for industrial bacterial strains. The bacteriophage exclusion (BREX) system is a recently described phage resistance system in prokaryotes. This work confirmed the function of the BREX system in L. casei and that the methyltransferase (pglX) is an indispensable part of the system. Overall, our study characterizes a BREX system component gene in lactic acid bacteria.

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Bacteriophage-encoded lytic enzymes control growth of contaminating Lactobacillus found in fuel ethanol fermentations.

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Bacteriophage-encoded lytic enzymes control growth of contaminating Lactobacillus found in fuel ethanol fermentations.

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