Comparative genomics of eight Lactobacillus buchneri strains isolated from food spoilage.

BACKGROUND: Lactobacillus buchneri is a lactic acid bacterium frequently associated with food bioprocessing and fermentation and has been found to be either beneficial or detrimental to industrial food processes depending on the application. The ability to metabolize lactic acid into acetic acid and 1,2-propandiol makes L. buchneri invaluable to the ensiling process, however, this metabolic activity leads to spoilage in other applications, and is especially damaging to the cucumber fermentation industry. This study aims to augment our genomic understanding of L. buchneri in order to make better use of the species in a wide range of applicable industrial settings. RESULTS: Whole-genome sequencing (WGS) was performed on seven phenotypically diverse strains isolated from spoiled, fermented cucumber and the ATCC type strain for L. buchneri, ATCC 4005. Here, we present our findings from the comparison of eight newly-sequenced and assembled genomes against two publicly available closed reference genomes, L. buchneri CD034 and NRRL B-30929. Overall, we see ~ 50% of all coding sequences are conserved across these ten strains. When these coding sequences are clustered by functional description, the strains appear to be enriched in mobile genetic elements, namely transposons. All isolates harbor at least one CRISPR-Cas system, and many contain putative prophage regions, some of which are targeted by the host's own DNA-encoded spacer sequences. CONCLUSIONS: Our findings provide new insights into the genomics of L. buchneri through whole genome sequencing and subsequent characterization of genomic features, building a platform for future studies and identifying elements for potential strain manipulation or engineering.

Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli.

The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming Lactobacillus species but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus, which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa.

Nitrogen utilization and metabolism in Ruminococcus albus 8.

The model rumen Firmicutes organism Ruminococcus albus 8 was grown using ammonia, urea, or peptides as the sole nitrogen source; growth was not observed with amino acids as the sole nitrogen source. Growth of R. albus 8 on ammonia and urea showed the same growth rate (0.08 h(-1)) and similar maximum cell densities (for ammonia, the optical density at 600 nm [OD600] was 1.01; and for urea, the OD600 was 0.99); however, growth on peptides resulted in a nearly identical growth rate (0.09 h(-1)) and a lower maximum cell density (OD600 = 0.58). To identify differences in gene expression and enzyme activities, the transcript abundances of 10 different genes involved in nitrogen metabolism and specific enzyme activities were analyzed by harvesting mRNA and crude protein from cells at the mid- and late exponential phases of growth on the different N sources. Transcript abundances and enzyme activities varied according to nitrogen source, ammonia concentration, and growth phase. Growth of R. albus 8 on ammonia and urea was similar, with the only observed difference being an increase in urease transcript abundance and enzyme activity in urea-grown cultures. Growth of R. albus 8 on peptides showed a different nitrogen metabolism pattern, with higher gene transcript abundance levels of gdhA, glnA, gltB, amtB, glnK, and ureC, as well as higher activities of glutamate dehydrogenase and urease. These results demonstrate that ammonia, urea, and peptides can all serve as nitrogen sources for R. albus and that nitrogen metabolism genes and enzyme activities of R. albus 8 are regulated by nitrogen source and the level of ammonia in the growth medium.

Addition of genes for cellobiase and pectinolytic activity in Escherichia coli for fuel ethanol production from pectin-rich lignocellulosic biomass.

Ethanologenic Escherichia coli strain KO11 was sequentially engineered to contain the Klebsiella oxytoca cellobiose phosphotransferase genes (casAB) as well as a pectate lyase (pelE) from Erwinia chrysanthemi, yielding strains LY40A (casAB) and JP07 (casAB pelE), respectively. To obtain an effective secretion of PelE, the Sec-dependent pathway out genes from E. chrysanthemi were provided on a cosmid to strain JP07 to construct strain JP07C. Finally, oligogalacturonide lyase (ogl) from E. chrysanthemi was added to produce strain JP08C. E. coli strains LY40A, JP07, JP07C, and JP08C possessed significant cellobiase activity in cell lysates, while only strains JP07C and JP08C demonstrated extracellular pectate lyase activity. Fermentations conducted by using a mixture of pure sugars representative of the composition of sugar beet pulp (SBP) showed that strains LY40A, JP07, JP07C, and JP08C were able to ferment cellobiose, resulting in increased ethanol production from 15 to 45% in comparison to that of KO11. Fermentations with SBP at very low fungal enzyme loads during saccharification revealed significantly higher levels of ethanol production for LY40A, JP07C, and JP08C than for KO11. JP07C ethanol yields were not considerably higher than those of LY40A; however, oligogalacturonide polymerization studies showed an increased breakdown of biomass to small-chain (degree of polymerization, ≤6) oligogalacturonides. JP08C achieved a further breakdown of polygalacturonate to monomeric sugars, resulting in a 164% increase in ethanol yields compared to those of KO11. The addition of commercial pectin methylesterase (PME) further increased JP08C ethanol production compared to that of LY40A by demethylating the pectin for enzymatic attack by pectin-degrading enzymes.

Characterization of two Paenibacillus amylolyticus strain 27C64 pectate lyases with activity on highly methylated pectin.

Two pectate lyases were identified from Paenibacillus amylolyticus 27C64; both enzymes demonstrated activity on methylated pectin in addition to polygalacturonic acid. PelA is in a subclass of the pectate lyase family III. PelB shows some features of pectate lyase family I but is highly divergent.

Klugiella xanthotipulae gen. nov., sp. nov., a novel member of the family Microbacteriaceae.

An actinobacterium, designated strain 44C3(T), was isolated in Michigan, USA, from the hindgut of the larvae of Tipula abdominalis, an aquatic crane fly, and was subjected to a polyphasic taxonomic investigation. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain represented a separate clade within the family Microbacteriaceae. It showed highest 16S rRNA gene sequence similarity with Cryobacterium psychrotolerans 0549(T) (96.5 %). Strain 44C3(T) had a novel B-type peptidoglycan. The peptidoglycan contained the diamino acid lysine, the peptide Gly-d-Glu was detected in the partial hydrolysate and alanine was the N terminus of the interpeptide bridge. No other amino acids found in other B-type peptidoglycans (including diaminobutyric acid, ornithine, homoserine and hydroxyglutamic acid) could be detected. The major menaquinones were MK-12 and MK-11, the major fatty acids were ai-C(15 : 0), ai-C(17 : 0) and i-C(16 : 0) and the DNA G+C content was 60.9 mol%. Analysis of the chemotaxonomic and phylogenetic data suggested that strain 44C3(T) represented a novel species of a new genus within the family Microbacteriaceae, for which the name Klugiella xanthotipulae gen. nov., sp. nov. is proposed. The type strain of Klugiella xanthotipulae is 44C3(T) (=DSM 18031(T) =ATCC BAA-1524(T)).