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.

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 ؋ 10 ؊2 ؎ 3.84 ؋ 10 ؊2 . This is the first direct documentation of conjugal transfer between gram-negative donor and gram-positive recipient bacteria in situ.Horizontal gene transfer between bacteria has gained considerable interest during the past 2 decades due to impacts on microbial adaptation to natural and anthropogenic stress and concerns related to the spreading of recombinant DNA. Bacteria are known to horizontally exchange genetic material by transformation, transduction, and conjugation (7). The mechanism of bacterial conjugation relies on exchange of mobile genetic elements (e.g., plasmids and conjugative transposons) and is believed to be one of the main mechanisms responsible for short-term microbial adaptation (34), transferring genetic material even among phylogenetically remote organisms (7, 21).Horizontal plasmid transfer has traditionally been studied by culture-dependent approaches, which are limited to the culturable fraction of environmental bacteria. However, since most bacteria are difficult to culture, with estimates that 1 to 5% of all bacteria are readily culturable (1,25,36), not only the transfer frequency, but also the host range of the plasmid may be grossly underestimated (34). The reluctant culturability of many soil bacteria requires that conjugative plasmid transfer be examined by cultivation-independent methods. Cultivationindependent approaches using fluorescent reporter genes (e.g., the green fluorescent protein gene, gfp) have previously been used to study the locations of transconjugants on the phylloplane (22, 23) and in biofilms (4, 11).The aim of the present study was to investigate in a cultivationindependent manner the host range and transfer frequency of a conjugative plasmid, pKJK10, among indigenous barley rhizosphere bacteria. To do this, we applied single-cell flow cytometry (FCM) analysis and cell sorting for cultivation-independent detection and quantification of plasmid transfer (32).The rhizosphere supports dense bacterial communities (20,24,33), and rhizosphere environments have been classified as hot spots for horizontal gene transfer (HGT) (7,17,20). Furthermore, the high cell density in the rhizosphere makes this environment well...

Mercury-resistant bacteria may be important players in mercury biogeochemistry. To assess the potential for mercury reduction by two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated natural conditions by using polycarbonate membranes as a growth support and a nonsterile soil slurry as a culture medium. Resistant bacteria were pregrown to microcolony-forming units (mCFU) before being plated on standard medium. Compared to direct plating, culturability was increased up to 2,800 times and numbers of mCFU were similar to the total number of mercury-resistant bacteria in the soils. Denaturing gradient gel electrophoresis analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the preincubation. A total of 25 different 16S rRNA gene sequences were observed, including Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. The diversity of isolates obtained by direct plating included eight different 16S rRNA gene sequences (Alpha-and Betaproteobacteria and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. With phylum-specific merA primers, PCR products were obtained for Alpha-and Betaproteobacteria and Actinobacteria but not for Bacteroidetes and Firmicutes. The similarity to known sequences ranged between 89 and 95%. One of the sequences did not result in a match in the BLAST search. The results illustrate the power of integrating advanced cultivation methodology with molecular techniques for the characterization of the diversity of mercury-resistant populations and assessing the potential for mercury reduction in contaminated environments.

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 b/c-Proteobacteria. b/c-Proteobacteria merA genes were successfully amplified from all soils, whereas Actinobacteria were amplified only from surface soil. merA clone libraries were constructed and sequenced. b/c-Proteobacteria sequences revealed high diversity in all soils, but limited vertical similarity. Less than 20% of the operational taxonomic units (OTU) (DNA sequences X95% identical) were shared between the different soils. Only one of the 62 OTU was X95% 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 a-Proteobacteria. An alignment of putative merA genes of genome sequenced mainly marine a-Proteobacteria was used for design of merA primers. PCR amplification of soil a-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.

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.

Quorum sensing enables bacteria to regulate expression of certain genes according to population density. N-acyl homoserine lactone (AHL)-based quorum sensing is known to be widespread among gram-negative bacteria. Several bacterial whole-cell biosensors for AHL detection have been developed and some were used in in situ studies of AHL production. From these studies our knowledge of the significance of quorum sensing in various environments has been improved. However, very little is known about production of AHLs in soil environments. In the present study, an approach for detecting AHL production in bulk soil was developed. A whole-cell biosensor based on the regulatory region of the lux-operon from Vibrio fischeri fused to gfp was constructed, resulting in a luxR-PluxI-gfpmut3*-fusion in the high copy plasmid, pAHL-GFP. Escherichia coli MC4100 harboring pAHL-GFP responded to the AHL-compound N-octanoyl homoserine lactone (OHL) by expressing green fluorescence. In situ application of E. coli MC4100/pAHL-GFP was tested by adding OHL in different concentrations to sterile soil microcosms. E. coli MC4100/pAHL-GFP were incubated in the soil microcosms and extracted by an improved Nycodenz-extraction method optimized for flow cytometry. The presence of induced cells was then verified by single-cell analysis by flow cytometry. OHL concentrations between 0.5 and 50 nmol per g soil were detected. When introducing the AHL-producing Serratia liquefaciens to soil microcosms, expression of green fluorescent protein was induced in E. coli MC4100/pAHL-GFP. Thereby, the ability of this strain to detect excretion of AHLs by S. liquefaciens in sterile soil was shown. The use of an improved extraction method and a whole-cell biosensor combined with flow cytometry analysis proved to be promising tools in future studies of AHL production by microbial populations in soil environments.

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.

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