Transient MutS-Based Hypermutation System for Adaptive Evolution of Lactobacillus casei to Low pH.

This study explored transient inactivation of the gene encoding the DNA mismatch repair enzyme MutS as a tool for adaptive evolution of Lactobacillus casei MutS deletion derivatives of L. casei 12A and ATCC 334 were constructed and subjected to a 100-day adaptive evolution process to increase lactic acid resistance at low pH. Wild-type parental strains were also subjected to this treatment. At the end of the process, the ΔmutS lesion was repaired in representative L. casei 12A and ATCC 334 ΔmutS mutant isolates. Growth studies in broth at pH 4.0 (titrated with lactic acid) showed that all four adapted strains grew more rapidly, to higher cell densities, and produced significantly more lactic acid than untreated wild-type cells. However, the adapted ΔmutS derivative mutants showed the greatest increases in growth and lactic acid production. Further characterization of the L. casei 12A-adapted ΔmutS derivative revealed that it had a significantly smaller cell volume, a rougher cell surface, and significantly better survival at pH 2.5 than parental L. casei 12A. Genome sequence analysis confirmed that transient mutS inactivation decreased DNA replication fidelity in both L. casei strains, and it identified genetic changes that might contribute to the lactic acid-resistant phenotypes of adapted cells. Targeted inactivation of three genes that had acquired nonsense mutations in the adapted L. casei 12A ΔmutS mutant derivative showed that NADH dehydrogenase (ndh), phosphate transport ATP-binding protein PstB (pstB), and two-component signal transduction system (TCS) quorum-sensing histidine protein kinase (hpk) genes act in combination to increase lactic acid resistance in L. casei 12A.IMPORTANCE Adaptive evolution has been applied to microorganisms to increase industrially desirable phenotypes, including acid resistance. We developed a method to increase the adaptability of Lactobacillus casei 12A and ATCC 334 through transient inactivation of the DNA mismatch repair enzyme MutS. Here, we show this method was effective in increasing the resistance of L. casei to lactic acid at low pH. Additionally, we identified three genes that contribute to increased acid resistance in L. casei 12A. These results provide valuable insight on methods to enhance an organism's fitness to complex phenotypes through adaptive evolution and targeted gene inactivation.

Mixed infections reveal virulence differences between host-specific bee pathogens.

Dynamics of host-pathogen interactions are complex, often influencing the ecology, evolution and behavior of both the host and pathogen. In the natural world, infections with multiple pathogens are common, yet due to their complexity, interactions can be difficult to predict and study. Mathematical models help facilitate our understanding of these evolutionary processes, but empirical data are needed to test model assumptions and predictions. We used two common theoretical models regarding mixed infections (superinfection and co-infection) to determine which model assumptions best described a group of fungal pathogens closely associated with bees. We tested three fungal species, Ascosphaera apis, Ascosphaera aggregata and Ascosphaera larvis, in two bee hosts (Apis mellifera and Megachile rotundata). Bee survival was not significantly different in mixed infections vs. solo infections with the most virulent pathogen for either host, but fungal growth within the host was significantly altered by mixed infections. In the host A. mellifera, only the most virulent pathogen was present in the host post-infection (indicating superinfective properties). In M. rotundata, the most virulent pathogen co-existed with the lesser-virulent one (indicating co-infective properties). We demonstrated that the competitive outcomes of mixed infections were host-specific, indicating strong host specificity among these fungal bee pathogens.

Detoxification and stress response genes expressed in a western North American bumble bee, Bombus huntii (Hymenoptera: Apidae).

BACKGROUND: The Hunt bumble bee (Bombus huntii Greene, Hymenoptera: Apidae) is a holometabolous, social insect important as a pollinator in natural and agricultural ecosystems in western North America. Bumble bees spend a significant amount of time foraging on a wide variety of flowering plants, and this activity exposes them to both plant toxins and pesticides, posing a threat to individual and colony survival. Little is known about what detoxification pathways are active in bumble bees, how the expression of detoxification genes changes across life stages, or how the number of detoxification genes expressed in B. huntii compares to other insects. RESULTS: We found B. huntii expressed at least 584 genes associated with detoxification and stress responses. The expression levels of some of these genes, such as those encoding the cytochrome P450s, glutathione S-transferases (GSTs) and glycosidases, vary among different life stages to a greater extent than do other genes. We also found that the number of P450s, GSTs and esterase genes expressed by B. huntii is similar to the number of these genes found in the genomes of other bees, namely Bombus terrestris, Bombus impatiens, Apis mellifera and Megachile rotundata, but many fewer than are found in the fly Drosophila melanogaster. CONCLUSIONS: Bombus huntii has transcripts for a large number of detoxification and stress related proteins, including oxidation and reduction enzymes, conjugation enzymes, hydrolytic enzymes, ABC transporters, cadherins, and heat shock proteins. The diversity of genes expressed within some detoxification pathways varies among the life stages and castes, and we typically identified more genes in the adult females than in larvae, pupae, or adult males, for most pathways. Meanwhile, we found the numbers of detoxification and stress genes expressed by B. huntii to be more similar to other bees than to the fruit fly. The low number of detoxification genes, first noted in the honey bee, appears to be a common phenomenon among bees, and perhaps results from their symbiotic relationship with plants. Many flowering plants benefit from pollinators, and thus offer these insects rewards (such as nectar) rather than defensive plant toxins.

The Streptococcus thermophilus protein Wzh functions as a phosphotyrosine phosphatase.

Amino acid residues that are important for metal binding and catalysis in Gram-positive phosphotyrosine phosphatases were identified in the Wzh protein of Streptococcus thermophilus MR-1C by using sequence comparisons. A His-tagged fusion Wzh protein was purified from Escherichia coli cultures and tested for phosphatase activity against synthetic phosphotyrosine and phosphoserine-threonine peptides. Purified Wzh released 2316.5 ± 138.7 pmol PO4·min(-1)·µg(-1) from phosphotyrosine peptide-1 and 2345.7 ± 135.2 pmol PO4·min(-1)·µg(-1) from phosphotyrosine peptide-2. The presence of the phosphotyrosine phosphatase inhibitor sodium vanadate decreased purified Wzh activity by 45%-50% at 1 mmol·L(-1), 74%-84% at 5 mmol·L(-1), and by at least 88% at 10 mmol·L(-1). Purified Wzh had no detectable activity against the phosphoserine-threonine peptide. These results clearly establish that S. thermophilus MR-1C Wzh functions as a phosphotyrosine phosphatase that could function to remove phosphate groups from proteins involved in exopolysaccharide biosynthesis, including the protein tyrosine kinase Wze and priming glycosyltransferase.

Intraspecific and interspecific interactions among proteins regulating exopolysaccharide synthesis in Streptococcus thermophilus, Streptococcus iniae, and Lactococcus lactis subsp. cremoris and the assessment of potential lateral gene transfer.

Using the yeast two-hybrid system, intraspecific protein interactions were detected in Streptococcus iniae and Lactococcus lactis subsp. cremoris between the transmembrane activation protein (CpsC and EpsA, respectively) and the protein tyrosine kinase (CpsD and EpsB, respectively), between two protein tyrosine kinases, and between the protein tyrosine kinase and the phosphotyrosine phosphatase (CpsB and EpsC, respectively). For each of these intraspecific interactions, interspecific interactions were also detected when one protein was from S. iniae and the other was from Streptococcus thermophilus . Interactions were also observed between two protein tyrosine kinases when one protein was from either of the Streptococcus species and the other from L. lactis subsp. cremoris. The results and sequence comparisons performed in this study support the conclusion that interactions among the components of the tyrosine kinase - phosphatase regulatory system are conserved in the order Lactobacillales and that interspecific genetic exchanges of the genes that encode these proteins have the potential to form functional recombinants. A better understanding of intraspecific and interspecific protein interactions involved in regulating exopolysaccharide biosynthesis may facilitate construction of improved strains for industrial uses as well as identification of factors needed to form functional regulatory complexes in naturally occurring recombinants.

Variation in Expression of Reference Genes across Life Stages of a Bee, Megachile rotundata.

The alfalfa leafcutting bee, Megachile rotundata is widely used in the western United States as a pollinator for alfalfa seed production. Unfortunately, immatures experience high mortality in agriculturally managed populations. Quantified gene expression could be used to identify how this bee responds during different life stages to pathogens, environmental toxins, and other stresses, but stably expressed reference genes are needed to normalize transcription data. We evaluated twelve candidate genes for their transcription stability across different life stages, including during and after diapause. RPS18 and RPL8 were the two most stably expressed genes, followed by RPS5 and RPL27A. These genes were also very stable even during and after diapause, while the most variable genes being APN, PMIIM, NPC2, and Cr-PII had increased expression levels during larval growth and were also variable during and after diapause. The four reference genes we identified in M. rotundata may prove useful for transcriptomic studies in other bees as well, such as honey bees.

Presence of Pathogen-killed Larvae Influences Nesting Behavior of the Alfalfa Leafcutting Bee (Hymenoptera: Megachilidae).

The alfalfa leafcutting bee (Megachile rotundata (Fabricius)), a commercial pollinator used for alfalfa seed production, is susceptible to chalkbrood disease via ingested fungal spores. Diseases of insects can elicit behavioral changes in their hosts, but there are no recorded behaviors of alfalfa leafcutting bees in response to this fungal exposure. We conducted field studies to determine whether bees in pathogen-dense environments altered their nesting patterns, specifically if bees exposed to fungal spores produced higher numbers of nest cells and whether the proportions of nest cells that failed as eggs or small larvae (a state known as 'pollen ball') were greater. We found that our control bees, nontreated bees which were not exposed to chalkbrood spores other than those in the natural environment, had the highest proportion of pollen ball cells. Bees experimentally exposed to infective spores created the lowest number of nests and the fewest cells. Bees experimentally exposed to heat killed noninfective spores produced the greatest number of nests and cells overall and the greatest number of healthy progeny. We conclude that there are underlying behaviors that are elicited in response to the presence of chalkbrood spores that reduce the proportion of failed nest cells (grooming) and increase retention of bees at nesting sites (delay of bee emergence). Through further study of these behaviors, bee managers can potentially increase the productivity of their bee populations.

Sequence analysis of plasmid pIR52-1 from Lactobacillus helveticus R0052 and investigation of its origin of replication.

Lactobacillus helveticus R0052 is a bacterium used in commercial probiotic preparations. R0052 contains a small, cryptic plasmid comprised of eight open reading frames, four of which encode proteins of unknown function. Based on the sequence of the replication initiation protein RepA, pIR52-1 is a member of the recently described RepA_N family of Gram-positive theta-replicating plasmids. The repA gene of pIR52-1 is the minimal origin of replication for L. helveticus and other Lactobacillus hosts. Additionally, pIR52-1 belongs to a subgroup of the RepA_N plasmid family which have RepA proteins of high amino acid identity and a conserved, non-coding element upstream of repA which, in pIR52-1, is responsible for the control of plasmid copy number and contributes to plasmid maintenance.

Transformation of Lactiplantibacillus plantarum and Apilactobacillus kunkeei is influenced by recipient cell growth temperature, vector replicon, and DNA methylation.

The effects of recipient cell growth temperature, vector choice, and DNA methylation on transformation efficiency were explored for Lactiplantibacillus plantarum strain B38 and Apilactobacillus kunkeei strains YH15 and 3L. All three parameters significantly affected transformation efficiency. L. plantarum B38 and A. kunkeei YH15 transformed at higher efficiencies with the pTW8 vector than with the pTRKH2 vector; conversely, A. kunkeei 3L transformed at higher efficiency with pTRKH2. Mean transformation efficiencies as high as 7.8 × 105 colony forming units (CFU) µg-1 were obtained with pTW8 for B38, as high as 1.2 × 105 CFU µg-1 with pTW8 for YH15, and as high as 3.4 × 106 CFU µg-1 with pTRKH2 for 3L. With respect to methylation, B38 and YH15 transformed at higher efficiencies with DNA that lacked dam methylation, while 3L transformed at higher efficiency with DNA that was dam methylated. Methylation at Escherichia coli dcm sites did not affect the ability of pTRKH2 or pTW8 to transform these strains. Recipient cell growth at 21 °C rather than at 37 °C significantly increased transformation efficiencies when using each strain's preferred vector and methylation state; pTW8 without dam methylation for B38 and YH15 and pTRKH2 with dam methylation for 3L.

Multiple pulse electroporation of lactic acid bacteria Lactococcus lactis and Lactobacillus casei.

Genetic manipulation of lactic acid bacteria is often difficult due to the inability to transform them with high efficiency. Multi-pulse electroporation offers a simple approach to increase transformation efficiencies. Using cells grown with 1% glycine and pretreated with lithium acetate and dithiothreitol, multi-pulse electroporation (five pulses of 12.5 kV cm-1) of Lactococcus lactis JB704 cells resulted in a transformation efficiency of up to 1.2 × 106 colony forming units (CFU) µg-1 pGK13, an 8-fold increase in the transformation efficiency compared to single pulse electroporation. Other cell growth and pretreatment conditions with JB704 resulted in lower transformation efficiencies but had 4-fold to 27-fold higher transformation efficiencies with the five pulse electroporations. With similarly grown and pretreated Lactobacillus casei 32G cells, multi-pulse electroporation (five pulses of 7.5 kV cm-1) resulted in a mean transformation efficiency of 7.3 × 103 CFU µg-1 pTRKH2, a 4-fold increase in the transformation efficiency compared to single pulse electroporation.

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

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.

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.

Complete Genome Sequence for Lactobacillus helveticus CNRZ 32, an Industrial Cheese Starter and Cheese Flavor Adjunct.

Lactobacillus helveticus is a lactic acid bacterium widely used in the manufacture of cheese and for production of bioactive peptides from milk proteins. We present the complete genome sequence for L. helveticus CNRZ 32, a strain particularly recognized for its ability to reduce bitterness and accelerate flavor development in cheese.

Polymorphic DNA sequences of the fungal honey bee pathogen Ascosphaera apis.

The pathogenic fungus Ascosphaera apis is ubiquitous in honey bee populations. We used the draft genome assembly of this pathogen to search for polymorphic intergenic loci that could be used to differentiate haplotypes. Primers were developed for five such loci, and the species specificities were verified using DNA from nine closely related species. The sequence variation was compared among 12 A. apis isolates at each of these loci, and two additional loci, the internal transcribed spacer of the ribosomal RNA (ITS) and a variable part of the elongation factor 1α (Ef1α). The degree of variation was then compared among the different loci, and three were found to have the greatest detection power for identifying A. apis haplotypes. The described loci can help to resolve strain differences and population genetic structures, to elucidate host-pathogen interaction and to test evolutionary hypotheses for the world's most important pollinator: the honey bee and one of its most common pathogens.

Identification of endopeptidase genes from the genomic sequence of Lactobacillus helveticus CNRZ32 and the role of these genes in hydrolysis of model bitter peptides.

Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5alpha derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, beta-casein (beta-CN) (f193-209) and alpha(S1)-casein (alpha(S1)-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10 degrees C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards alpha(S1)-CN (f1-9) and beta-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze beta-CN (f193-209) and alpha(S1)-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides alpha(S1)-CN (f1-9), alpha(S1)-CN (f1-13), and alpha(S1)-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with beta-CN (f193-209) and alpha(S1)-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.