Versatile Lactococcus lactis strains improve texture in both fermented milk and soybean matrices.

Lactic acid bacteria (LAB) have long been used to extend the shelf life and improve the taste and texture of fermented milk. In this study, we investigated the texturing potential of LAB in plant-based fermentation by high-throughput screening of 1232 Lactococcus lactis strains for texture in milk and liquid soybean matrices. We found that most strains with texturing abilities in fermented milk were also capable of enhancing the texture in fermented soybean, despite the large differences in composition of the two matrices. Exocellular polysaccharide production is believed to contribute positively to fermented milk and plant-base texture. It appeared as if it was the properties of the polysaccharides rather than their protein interaction partners that were responsible for the enhanced texture in both matrices. We mined whole genome sequences of texturing strains for polysaccharide biosynthesis (eps) gene clusters. The comparative genomics approach revealed 10 texturing strains with novel eps gene clusters. Currently, the relationship between the novel genes and their functionality in milk and plant matrices is unknown.

Biofilm cultivation facilitates coexistence and adaptive evolution in an industrial bacterial community.

The majority of ecological, industrial and medical impacts of bacteria result from diverse communities containing multiple species. This diversity presents a significant challenge as co-cultivation of multiple bacterial species frequently leads to species being outcompeted and, with this, the possibility to manipulate, evolve and improve bacterial communities is lost. Ecological theory predicts that a solution to this problem will be to grow species in structured environments, which reduces the likelihood of competitive exclusion. Here, we explored the ability of cultivation in a structured environment to facilitate coexistence, evolution, and adaptation in an industrially important community: Lactococcus lactis and Leuconostoc mesenteroides frequently used as dairy starter cultures. As commonly occurs, passaging of these two species together in a liquid culture model led to the loss of one species in 6 of 20 lineages (30%). By contrast, when we co-cultured the two species as biofilms on beads, a stable coexistence was observed in all lineages studied for over 100 generations. Moreover, we show that the co-culture drove evolution of new high-yield variants, which compared to the ancestor grew more slowly, yielded more cells and had enhanced capability of biofilm formation. Importantly, we also show that these high-yield biofilm strains did not evolve when each species was passaged in monoculture in the biofilm model. Therefore, both co-culture and the biofilm model were conditional for these high-yield strains to evolve. Our study underlines the power of ecological thinking-namely, the importance of structured environments for coexistence-to facilitate cultivation, evolution, and adaptation of industrially important bacterial communities.

Gene-Trait Matching and Prevalence of Nisin Tolerance Systems in Lactococus lactis.

Lactococcus lactis cheese starter cultures typically contain a mix of many strains and may include variants that produce and/or tolerate the antimicrobial bacteriocin nisin. Nisin is well-established as an effective agent against several undesirable Gram-positive bacteria in cheese and various other foods. In the current study, we have examined the effect of nisin on 710 individual L. lactis strains during milk fermentations. Changes in milk acidification profiles with and without nisin exposure, ranging from unaltered acidification to loss of acidification, could be largely explained by the type(s) and variants of nisin immunity and nisin degradation genes present, but surprisingly, also by genotypic lineage (L. lactis ssp. cremoris vs. ssp. lactis). Importantly, we identify that nisin degradation by NSR is frequent among L. lactis and therefore likely the main mechanism by which dairy-associated L. lactis strains tolerate nisin. Insights from this study on the strain-specific effect of nisin tolerance and degradation during milk acidification is expected to aid in the design of nisin-compatible cheese starter cultures.

Machine learning predicts and provides insights into milk acidification rates of Lactococcus lactis.

Lactococcus lactis strains are important components in industrial starter cultures for cheese manufacturing. They have many strain-dependent properties, which affect the final product. Here, we explored the use of machine learning to create systematic, high-throughput screening methods for these properties. Fast acidification of milk is such a strain-dependent property. To predict the maximum hourly acidification rate (Vmax), we trained Random Forest (RF) models on four different genomic representations: Presence/absence of gene families, counts of Pfam domains, the 8 nucleotide long subsequences of their DNA (8-mers), and the 9 nucleotide long subsequences of their DNA (9-mers). Vmax was measured at different temperatures, volumes, and in the presence or absence of yeast extract. These conditions were added as features in each RF model. The four models were trained on 257 strains, and the correlation between the measured Vmax and the predicted Vmax was evaluated with Pearson Correlation Coefficients (PC) on a separate dataset of 85 strains. The models all had high PC scores: 0.83 (gene presence/absence model), 0.84 (Pfam domain model), 0.76 (8-mer model), and 0.85 (9-mer model). The models all based their predictions on relevant genetic features and showed consensus on systems for lactose metabolism, degradation of casein, and pH stress response. Each model also predicted a set of features not found by the other models.

High-throughput screening for texturing Lactococcus strains.

In the food industry, lactic acid bacteria (LAB) are used in dairy fermentations, extending the shelf life by lowering the pH and also affecting taste and texture of the fermented milk. The texture of fermented milk is an important quality parameter, affecting consumer acceptance. Finding LAB providing desired texture of a product is time consuming and laborious when using standard methods for measuring texture, e.g. rheology measurements. Screening of 986 Lactococcus lactis strains resulted in few strains with the ability to enhance texture, demonstrating the necessity of implementation of high-throughput screening methods. A high-throughput screening assay was developed, combining small-scale 96-well microtiter plates and pressure measurements during liquid handling, e.g. aspiration, to find strains that give good texture in fermented milk. Only about 1% of the strains were found to enhance milk texture. Two of the texturing strains belong to L. lactis subsp. lactis, which are the first texturing strains from this subsp. reported. Mining for eps gene clusters responsible for exocellular polysaccharide production was performed, as polysaccharide production can contribute positively to fermented milk texture. Comparative genomics approach revealed four types of texturing L. lactis strains with diverse eps gene clusters.

Polysaccharide production by lactic acid bacteria: from genes to industrial applications.

The ability to produce polysaccharides with diverse biological functions is widespread in bacteria. In lactic acid bacteria (LAB), production of polysaccharides has long been associated with the technological, functional and health-promoting benefits of these microorganisms. In particular, the capsular polysaccharides and exopolysaccharides have been implicated in modulation of the rheological properties of fermented products. For this reason, screening and selection of exocellular polysaccharide-producing LAB has been extensively carried out by academia and industry. To further exploit the ability of LAB to produce polysaccharides, an in-depth understanding of their biochemistry, genetics, biosynthetic pathways, regulation and structure-function relationships is mandatory. Here, we provide a critical overview of the latest advances in the field of glycosciences in LAB. Surprisingly, the understanding of the molecular processes involved in polysaccharide synthesis is lagging behind, and has not accompanied the increasing commercial value and application potential of these polymers. Seizing the natural diversity of polysaccharides for exciting new applications will require a concerted effort encompassing in-depth physiological characterization of LAB at the systems level. Combining high-throughput experimentation with computational approaches, biochemical and structural characterization of the polysaccharides and understanding of the structure-function-application relationships is essential to achieve this ambitious goal.

Construction and use of flow cytometry optimized plasmid-sensor strains.

Determining the stability of plasmids in bacterial populations is traditionally performed by isolating a large number of clones followed by screening for the presence of plasmids by replica transfer to plasmid-selective agar plates. This is often a laborious task, especially when the intrinsic stability of the plasmid is high. The method presented here relies on a phenotypic (green fluorescence protein) marker, which is switched on if the host bacteria loses the residing plasmid. The incorporation of flow cytometry for single-cell detection and discrimination between plasmid-free and plasmid-harboring cells in a bacterial population facilitates a very high throughput of cells and thus provides excellent sensitivity and statistics toward detecting even very low levels of plasmid instability.

Acclimation of subsurface microbial communities to mercury.

We studied the acclimation to mercury of bacterial communities of different depths from contaminated and noncontaminated floodplain soils. The level of mercury tolerance of the bacterial communities from the contaminated site was higher than those of the reference site. Furthermore, the level of mercury tolerance and functional versatility of bacterial communities in contaminated soils initially were higher for surface soil, compared with the deeper soils. However, following new mercury exposure, no differences between bacterial communities were observed, which indicates a high adaptive potential of the subsurface communities, possibly due to differences in the availability of mercury. IncP-1 trfA genes were detected in extracted community DNA from all soil depths of the contaminated site, and this finding was correlated to the isolation of four different mercury-resistance plasmids, all belonging to the IncP-1beta group. The abundance of merA and IncP-1 plasmid carrying populations increased, after new mercury exposure, which could be the result of selection as well as horizontal gene exchange. The data in this study suggest a role for IncP-1 plasmids in the acclimation to mercury of surface as well as subsurface soil microbial communities.

High diversity of bacterial mercuric reductase genes from surface and sub-surface floodplain soil (Oak Ridge, USA).

DNA was extracted from different depth soils (0-5, 45-55 and 90-100 cm below surface) sampled at Lower East Fork Poplar Creek floodplain (LEFPCF), Oak Ridge (TN, USA). The presence of merA genes, encoding the mercuric reductase, the key enzyme in detoxification of mercury in bacteria, was examined by PCR targeting Actinobacteria, Firmicutes or beta/gamma-Proteobacteria. beta/gamma-Proteobacteria merA genes were successfully amplified from all soils, whereas Actinobacteria were amplified only from surface soil. merA clone libraries were constructed and sequenced. beta/gamma-Proteobacteria sequences revealed high diversity in all soils, but limited vertical similarity. Less than 20% of the operational taxonomic units (OTU) (DNA sequences > or = 95% identical) were shared between the different soils. Only one of the 62 OTU was > or = 95% identical to a GenBank sequence, highlighting that cultivated bacteria are not representative of what is found in nature. Fewer merA sequences were obtained from the Actinobacteria, but these were also diverse, and all were different from GenBank sequences. A single clone was most closely related to merA of alpha-Proteobacteria. An alignment of putative merA genes of genome sequenced mainly marine alpha-Proteobacteria was used for design of merA primers. PCR amplification of soil alpha-Proteobacteria isolates and sequencing revealed that they were very different from the genome-sequenced bacteria (only 62%-66% identical at the amino-acid level), although internally similar. In light of the high functional diversity of mercury resistance genes and the limited vertical distribution of shared OTU, we discuss the role of horizontal gene transfer as a mechanism of bacterial adaptation to mercury.

Cultivation-independent examination of horizontal transfer and host range of an IncP-1 plasmid among gram-positive and gram-negative bacteria indigenous to the barley rhizosphere.

The host range and transfer frequency of an IncP-1 plasmid (pKJK10) among indigenous bacteria in the barley rhizosphere was investigated. A new flow cytometry-based cultivation-independent method for enumeration and sorting of transconjugants for subsequent 16S rRNA gene classification was used. Indigenous transconjugant rhizosphere bacteria were collected by fluorescence-activated cell sorting and identified by cloning and sequencing of 16S rRNA genes from the sorted cells. The host range of the pKJK10 plasmid was exceptionally broad, as it included not only bacteria belonging to the alpha, beta, and gamma subclasses of the Proteobacteria, but also Arthrobacter sp., a gram-positive member of the Actinobacteria. The transfer frequency (transconjugants per donor) from the Pseudomonas putida donor to the indigenous bacteria was 7.03 x 10(-2) +/- 3.84 x 10(-2). This is the first direct documentation of conjugal transfer between gram-negative donor and gram-positive recipient bacteria in situ.

Direct detection and quantification of horizontal gene transfer by using flow cytometry and gfp as a reporter gene.

A new cultivation-independent method for studying conjugal gene transfer between bacteria was evaluated. The method was based on direct detection and enumeration of donor and transconjugant bacterial cells by flow cytometry. Specific detection of transconjugants was obtained by using a conjugative plasmid tagged with a reporter gene (gfp) encoding green fluorescent protein. A chromosomal encoded repressor (lacI(ql)) repressed expression of GFP in the donor bacteria. Enumeration of the donor cells was performed after induction of GFP expression by the addition of inducer isopropyl-thio-beta-D-galactoside (IPTG). The method presented here provided simple and precise quantification of horizontal gene transfer between both Escherichia coli and Pseudomonas putida strains.