Jeff R. Broadbent

Comparative genomics of the lactic acid bacteria

Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.

Genome sequence and comparative genome analysis of Lactobacillus casei: insights into their niche-associated evolution.

Lactobacillus casei is remarkably adaptable to diverse habitats and widely used in the food industry. To reveal the genomic features that contribute to its broad ecological adaptability and examine the evolution of the species, the genome sequence of L. casei ATCC 334 is analyzed and compared with other sequenced lactobacilli. This analysis reveals that ATCC 334 contains a high number of coding sequences involved in carbohydrate utilization and transcriptional regulation, reflecting its requirement for dealing with diverse environmental conditions. A comparison of the genome sequences of ATCC 334 to L. casei BL23 reveals 12 and 19 genomic islands, respectively. For a broader assessment of the genetic variability within L. casei, gene content of 21 L. casei strains isolated from various habitats (cheeses, n = 7; plant materials, n = 8; and human sources, n = 6) was examined by comparative genome hybridization with an ATCC 334-based microarray. This analysis resulted in identification of 25 hypervariable regions. One of these regions contains an overrepresentation of genes involved in carbohydrate utilization and transcriptional regulation and was thus proposed as a lifestyle adaptation island. Differences in L. casei genome inventory reveal both gene gain and gene decay. Gene gain, via acquisition of genomic islands, likely confers a fitness benefit in specific habitats. Gene decay, that is, loss of unnecessary ancestral traits, is observed in the cheese isolates and likely results in enhanced fitness in the dairy niche. This study gives the first picture of the stable versus variable regions in L. casei and provides valuable insights into evolution, lifestyle adaptation, and metabolic diversity of L. casei.

Physiological and Transcriptional Response of Lactobacillus casei ATCC 334 to Acid Stress

This study investigated features of the acid tolerance response (ATR) in Lactobacillus casei ATCC 334. To optimize ATR induction, cells were acid adapted for 10 or 20 min at different pH values (range, 3.0 to 5.0) and then acid challenged at pH 2.0. Adaptation over a broad range of pHs improved acid tolerance, but the highest survival was noted in cells acid adapted for 10 or 20 min at pH 4.5. Analysis of cytoplasmic membrane fatty acids (CMFAs) in acid-adapted cells showed that they had significantly (P < 0.05) higher total percentages of saturated and cyclopropane fatty acids than did control cells. Specifically, large increases in the percentages of C(14:0), C(16:1n(9)), C(16:0), and C(19:0(11c)) were noted in the CMFAs of acid-adapted and acid-adapted, acid-challenged cells, while C(18:1n(9)) and C(18:1n(11)) showed the greatest decrease. Comparison of the transcriptome from control cells (grown at pH 6.0) against that from cells acid adapted for 20 min at pH 4.5 indicated that acid adaption invoked a stringent-type response that was accompanied by other functions which likely helped these cells resist acid damage, including malolactic fermentation and intracellular accumulation of His. Validation of microarray data was provided by experiments that showed that L. casei survival at pH 2.5 was improved at least 100-fold by chemical induction of the stringent response or by the addition of 30 mM malate or 30 mM histidine to the acid challenge medium. To our knowledge, this is the first report that intracellular histidine accumulation may be involved in bacterial acid resistance.

Analysis of the Lactobacillus casei supragenome and its influence in species evolution and lifestyle adaptation.

BackgroundThe broad ecological distribution of L. casei makes it an insightful subject for research on genome evolution and lifestyle adaptation. To explore evolutionary mechanisms that determine genomic diversity of L. casei, we performed comparative analysis of 17 L. casei genomes representing strains collected from dairy, plant, and human sources.ResultsDifferences in L. casei genome inventory revealed an open pan-genome comprised of 1,715 core and 4,220 accessory genes. Extrapolation of pan-genome data indicates L. casei has a supragenome approximately 3.2 times larger than the average genome of individual strains. Evidence suggests horizontal gene transfer from other bacterial species, particularly lactobacilli, has been important in adaptation of L. casei to new habitats and lifestyles, but evolution of dairy niche specialists also appears to involve gene decay.ConclusionsGenome diversity in L. casei has evolved through gene acquisition and decay. Acquisition of foreign genomic islands likely confers a fitness benefit in specific habitats, notably plant-associated niches. Loss of unnecessary ancestral traits in strains collected from bacterial-ripened cheeses supports the hypothesis that gene decay contributes to enhanced fitness in that niche. This study gives the first evidence for a L. casei supragenome and provides valuable insights into mechanisms for genome evolution and lifestyle adaptation of this ecologically flexible and industrially important lactic acid bacterium. Additionally, our data confirm the Distributed Genome Hypothesis extends to non-pathogenic, ecologically flexible species like L. casei.

Comparative genomics and transcriptional analysis of prophages identified in the genomes of Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei.

ABSTRACT Lactobacillus gasseri ATCC 33323, Lactobacillus salivarius subsp. salivarius UCC 118, and Lactobacillus casei ATCC 334 contain one (LgaI), four (Sal1, Sal2, Sal3, Sal4), and one (Lca1) distinguishable prophage sequences, respectively. Sequence analysis revealed that LgaI, Lca1, Sal1, and Sal2 prophages belong to the group of Sfi11-like pac site and cos site Siphoviridae, respectively. Phylogenetic investigation of these newly described prophage sequences revealed that they have not followed an evolutionary development similar to that of their bacterial hosts and that they show a high degree of diversity, even within a species. The attachment sites were determined for all these prophage elements; LgaI as well as Sal1 integrates in tRNA genes, while prophage Sal2 integrates in a predicted arginino-succinate lyase-encoding gene. In contrast, Lca1 and the Sal3 and Sal4 prophage remnants are integrated in noncoding regions in the L. casei ATCC 334 and L. salivarius UCC 118 genomes. Northern analysis showed that large parts of the prophage genomes are transcriptionally silent and that transcription is limited to genome segments located near the attachment site. Finally, pulsed-field gel electrophoresis followed by Southern blot hybridization with specific prophage probes indicates that these prophage sequences are narrowly distributed within lactobacilli.

Phenotypic and genotypic analysis of amino acid auxotrophy in Lactobacillus helveticus CNRZ 32.

ABSTRACT The conversion of amino acids into volatile and nonvolatile compounds by lactic acid bacteria in cheese is thought to represent the rate-limiting step in the development of mature flavor and aroma. Because amino acid breakdown by microbes often entails the reversible action of enzymes involved in biosynthetic pathways, our group investigated the genetics of amino acid biosynthesis in Lactobacillus helveticus CNRZ 32, a commercial cheese flavor adjunct that reduces bitterness and intensifies flavor notes. Most lactic acid bacteria are auxotrophic for several amino acids, and L. helveticus CNRZ 32 requires 14 amino acids. The reconstruction of amino acid biosynthetic pathways from a draft-quality genome sequence for L. helveticus CNRZ 32 revealed that amino acid auxotrophy in this species was due primarily to gene absence rather than point mutations, insertions, or small deletions, with good agreement between gene content and phenotypic amino acid requirements. One exception involved the phenotypic requirement for Asp (or Asn), which genome predictions suggested could be alleviated by citrate catabolism. This prediction was confirmed by the growth of L. helveticus CNRZ 32 after the addition of citrate to a chemically defined medium that lacked Asp and Asn. Genome analysis also predicted that L. helveticus CNRZ 32 possessed ornithine decarboxylase activity and would therefore catalyze the conversion of ornithine to putrescine, a volatile biogenic amine. However, experiments to confirm ornithine decarboxylase activity in L. helveticus CNRZ 32 by the use of several methods were unsuccessful, which indicated that this bacterium likely does not contribute to putrescine production in cheese.

Broad scope method for creating humanized animal models for animal health and disease research through antibiotic treatment and human fecal transfer

Traditionally, mouse humanization studies have used human fecal transfer to germ-free animals. This practice requires gnotobiotic facilities and is restricted to gnotobiotic mouse lines, which limits humanized mouse research. We have developed a generalizable method to humanize non germ-free mice using antibiotic treatment and human fecal transfer. The method involves depleting resident intestinal microbiota with broad-spectrum antibiotics, introducing human microbiota from frozen fecal samples by weekly gavage, and maintaining mice in HEPA-filtered microisolator cages. Pyrosequencing cecal microbiota 16S rRNA genes showed that recipient mice adopt a humanized microbiota profile analogous to their human donors, and distinct from mice treated with only antibiotics (no fecal transfer) or untreated control mice. In the humanized mice, 75% of the sequence mass was observed in their respective human donor and conversely, 68% of the donor sequence mass was recovered in the recipient mice. Principal component analyses of GC- and HPLC-separated cecal metabolites were performed to determine effects of transplanted microbiota on the metabolome. Cecal metabolite profiles of mice treated with only antibiotics (no fecal transfer) and control mice were dissimilar from each other and from humanized mice. Metabolite profiles for mice humanized from different donor samples clustered near each other, yet were sufficiently distinct that separate clusters were apparent for each donor. Also, cecal concentrations of 57 metabolites were significantly different between humanization treatments. These data demonstrate that our protocol can be used to humanize non germ-free mice and is sufficiently robust to generate metabolomic differences between mice humanized from different human donors.

Genome –Scale Reconstruction of Metabolic Networks of Lactobacillus casei ATCC 334 and 12A

Lactobacillus casei strains are widely used in industry and the utility of this organism in these industrial applications is strain dependent. Hence, tools capable of predicting strain specific phenotypes would have utility in the selection of strains for specific industrial processes. Genome-scale metabolic models can be utilized to better understand genotype-phenotype relationships and to compare different organisms. To assist in the selection and development of strains with enhanced industrial utility, genome-scale models for L. casei ATCC 334, a well characterized strain, and strain 12A, a corn silage isolate, were constructed. Draft models were generated from RAST genome annotations using the Model SEED database and refined by evaluating ATP generating cycles, mass-and-charge-balances of reactions, and growth phenotypes. After the validation process was finished, we compared the metabolic networks of these two strains to identify metabolic, genetic and ortholog differences that may lead to different phenotypic behaviors. We conclude that the metabolic capabilities of the two networks are highly similar. The L. casei ATCC 334 model accounts for 1,040 reactions, 959 metabolites and 548 genes, while the L. casei 12A model accounts for 1,076 reactions, 979 metabolites and 640 genes. The developed L. casei ATCC 334 and 12A metabolic models will enable better understanding of the physiology of these organisms and be valuable tools in the development and selection of strains with enhanced utility in a variety of industrial applications.

Identification of plasmalogens in the cytoplasmic membrane of Bifidobacterium animalis subsp. lactis

ABSTRACT Plasmalogens are ether-linked lipids that may influence oxidative stress resistance of eukaryotic cell membranes. Since bacterial membrane composition can influence environmental stress resistance, we explored the prevalence of plasmalogens in the cytoplasmic membrane of Bifidobacterium animalis subsp. lactis. Results showed plasmalogens are a major component of the B. animalis subsp. lactis membrane.

High efficiency electrotransformation of Lactobacillus casei

We investigated whether protocols allowing high efficiency electrotransformation of other lactic acid bacteria were applicable to five strains of Lactobacillus casei (12A, 32G, A2-362, ATCC 334 and BL23). Addition of 1% glycine or 0.9 M NaCl during cell growth, limitation of the growth of the cell cultures to OD600 0.6-0.8, pre-electroporation treatment of cells with water or with a lithium acetate (100 mM)/dithiothreitol (10 mM) solution and optimization of electroporation conditions all improved transformation efficiencies. However, the five strains varied in their responses to these treatments. Transformation efficiencies of 10(6) colony forming units μg(-1) pTRKH2 DNA and higher were obtained with three strains which is sufficient for construction of chromosomal gene knock-outs and gene replacements.