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Lactococcus lactis subsp. lactis KF147 as a non-dairy strain from lactic acid bacteria (LAB) can inhabit plant tissues. It can grow on complex carbohydrates derived from plant cell walls. Its genome size is one of the largest among the sequenced lactococcal strains, possessing many genes that do not have homologues in the published genome sequences of dairy-associated L. lactis strains. In silico analysis has identified a gene cluster encoding a hybrid NRPS-PKS system (composed of non-ribosomal peptide synthetases and polyketide synthases) in the L. lactis KF147 genome, as first example of a LAB possessing such hybrid mega-enzymes. Hybrid systems produce hybrid NRP-PK secondary metabolites (natural products) in a wide variety of bacteria, fungi, and plants. In the hybrid NRPS-PKS system of L. lactis KF147, a total of 21 NRPS and 8 PKS domains were identified that are arranged into 6 NRPS modules, 3 PKS modules, and two single functional domains (trans-acyl-transferase "transAT" and thioesterase). We found homologous hybrid systems having similar gene, module, and domain organization in six other L. lactis strains and 25 strains of the dental cariogenic Streptococcus mutans. This study mainly aimed to predict the structure and function of the hybrid NRP-PK product of L. lactis KF147 using comparative genomics techniques, and included a detailed analysis of the regulatory system. Various bioinformatical approaches were used to predict the substrate specificity of the six A domains and the iterative transAT domain. Functional conservation of the A domains within different-niche-associated strains supported the prediction of the primary core structure of the putative hybrid natural product to be Leu-DLeu-Asp-DAsn-Gly-MC-MC-MC-DAsp (MC = Malonyl-CoA). Oxidative stress resistance and biofilm formation are the most probable functions of this hybrid system. The need for such a system in two different niches is argued, as an adaptation of L. lactis and S. mutans to adhere to plant tissues and human teeth, respectively, in an oxidative environment. ispartof: Current Microbiology vol:77 issue:1 pages:136-145 ispartof: location:United States status: published

The lactic acid bacterium Lactococcus lactis is widely used for the fermentation of dairy products. Here, we present the draft genome sequences of 11 L. lactis subsp. cremoris strains isolated from different environments.

Whole-genome sequencing and phenotypic testing of 104 strains of Bacillus licheniformis and Bacillus paralicheniformis from a variety of sources and time periods was used to characterize the genetic background and evolution of (putative) antimicrobial resistance mechanisms. Core proteins were identified in draft genomes and a phylogenetic analysis based on single amino acid polymorphisms allowed the species to be separated into two phylogenetically distinct clades with one outlier. Putative antimicrobial resistance genes were identified and mapped. A chromosomal ermD gene was found at the same location in all B. paralichenformis and in 27% of B. licheniformis genomes. Erythromycin resistance correlated very well with the presence of ermD. The putative streptomycin resistance genes, aph and aadK, were found in the chromosome of all strains as adjacent loci. Variations in amino acid sequence did not correlate with streptomycin susceptibility although the species were less susceptible than other Bacillus species. A putative chloramphenicol resistance gene (cat), encoding a novel chloramphenicol acetyltransferase protein was also found in the chromosome of all strains. Strains encoding a truncated CAT protein were sensitive to chloramphenicol. For all four resistance genes, the diversity and genetic context followed the overall phylogenetic relationship. No potentially mobile genetic elements were detected in their vicinity. Moreover, the genes were only distantly related to previously-described cat, aph, aad and erm genes present on mobile genetic elements or in other species. Thus, these genes are suggested to be intrinsic to B. licheniformis and B. paralicheniformis and part of their ancient resistomes. Since there is no evidence supporting horizontal transmission, these genes are not expected to add to the pool of antibiotic resistance elements considered to pose a risk to human or animal health. Whole-genome based phylogenetic and sequence analysis, combined with phenotypic testing, is proposed to be suitable for determining intrinsic resistance and evolutionary relationships.

Gluten are proline- and glutamine-rich proteins present in wheat, barley, and rye and contain the immunogenic sequences that drive celiac disease (CD). Rothia mucilaginosa, an oral microbial colonizer, can cleave these gluten epitopes. The aim was to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for CD. The membrane-associated R. mucilaginosa proteins were extracted and separated by DEAE chromatography. Enzyme activities were monitored with paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography. Epitope elimination was determined in R5 and G12 ELISAs. The gliadin-degrading Rothia enzymes were identified by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC), and ROTMU0001_240 (C6R5V8_9MICC). A search with the Basic Local Alignment Search Tool revealed that these are subtilisin-like serine proteases belonging to the peptidase S8 family. Alignment of the major Rothia subtilisins indicated that all contain the catalytic triad with Asp (D), His (H), and Ser (S) in the D-H-S order. They cleaved succinyl-Ala-Ala-Pro-Phe-paranitroanilide, a substrate for subtilisin with Pro in the P2 position, as in Tyr-Pro-Gln and Leu-Pro-Tyr in gluten, which are also cleaved. Consistently, FRET substrates of gliadin immunogenic epitopes comprising Xaa-Pro-Xaa motives were rapidly hydrolyzed. The Rothia subtilisins and two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase, efficiently degraded the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 and G12 antibodies. This study identified Rothia and food-grade Bacillus subtilisins as promising new candidates for enzyme therapeutics in CD.

The lactic acid bacterium Lactococcus lactis is widely used for the production of fermented dairy products. Here, we present the draft genome sequences of 24 L. lactis strains isolated from different environments and geographic locations.

The lactic acid bacterium Lactococcus lactis is widely used for the fermentation of dairy products. Here, we present the draft genome sequences of 11 L. lactis subsp. cremoris strains isolated from different environments.

The lactic acid bacterium Lactococcus lactis is widely used for the production of fermented dairy products. Here, we present the draft genome sequences of 24 L. lactis strains isolated from different environments and geographic locations.

Enterococcus faecium NRRL B-2354 is a surrogate microorganism used in place of pathogens for validation of thermal processing technologies and systems. We evaluated the safety of strain NRRL B-2354 based on its genomic and functional characteristics. The genome of E. faecium NRRL B-2354 was sequenced and found to comprise a 2,635,572-bp chromosome and a 214,319-bp megaplasmid. A total of 2,639 coding sequences were identified, including 45 genes unique to this strain. Hierarchical clustering of the NRRL B-2354 genome with 126 other E. faecium genomes as well as pbp5 locus comparisons and multilocus sequence typing (MLST) showed that the genotype of this strain is most similar to commensal, or community-associated, strains of this species. E. faecium NRRL B-2354 lacks antibiotic resistance genes, and both NRRL B-2354 and its clonal relative ATCC 8459 are sensitive to clinically relevant antibiotics. This organism also lacks, or contains nonfunctional copies of, enterococcal virulence genes including acm , cyl , the ebp operon, esp , gelE , hyl , IS 16 , and associated phenotypes. It does contain scm , sagA , efaA , and pilA , although either these genes were not expressed or their roles in enterococcal virulence are not well understood. Compared with the clinical strains TX0082 and 1,231,502, E. faecium NRRL B-2354 was more resistant to acidic conditions (pH 2.4) and high temperatures (60°C) and was able to grow in 8% ethanol. These findings support the continued use of E. faecium NRRL B-2354 in thermal process validation of food products.

Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmid-free and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC). The copper resistance could be used as a marker for the conjugation of pNZ712 to L. lactis MG1614. A genome comparison with the plasmid cured daughter strain MG1363 showed that the number of single nucleotide polymorphisms that accumulated in the laboratory since the strains diverted more than 30 years ago is limited to 11 of which only 5 lead to amino acid changes. The 16-kb plasmid pSH74 was found to contain a novel 8-kb pilus gene cluster spaCB-spaA-srtC1-srtC2, which is predicted to encode a pilin tip protein SpaC, a pilus basal subunit SpaB, and a pilus backbone protein SpaA. The sortases SrtC1/SrtC2 are most likely involved in pilus polymerization while the chromosomally encoded SrtA could act to anchor the pilus to peptidoglycan in the cell wall. Overexpression of the pilus gene cluster from a multi-copy plasmid in L. lactis MG1363 resulted in cell chaining, aggregation, rapid sedimentation and increased conjugation efficiency of the cells. Electron microscopy showed that the over-expression of the pilus gene cluster leads to appendices on the cell surfaces. A deletion of the gene encoding the putative basal protein spaB, by truncating spaCB, led to more pilus-like structures on the cell surface, but cell aggregation and cell chaining were no longer observed. This is consistent with the prediction that spaB is involved in the anchoring of the pili to the cell.

Saccharomyces cerevisiae cannot metabolize d-galacturonate, an important monomer of pectin. Use of S. cerevisiae for production of ethanol or other compounds of interest from pectin-rich feedstocks therefore requires introduction of a heterologous pathway for d-galacturonate metabolism. Bacterial d-galacturonate pathways involve d-galacturonate isomerase, d-tagaturonate reductase and three additional enzymes. This study focuses on functional expression of bacterial d-galacturonate isomerases in S. cerevisiae. After demonstrating high-level functional expression of a d-tagaturonate reductase gene (uxaB from Lactococcus lactis), the resulting yeast strain was used to screen for functional expression of six codon-optimized bacterial d-galacturonate isomerase (uxaC) genes. The L. lactis uxaC gene stood out, yielding a tenfold higher enzyme activity than the other uxaC genes. Efficient expression of d-galacturonate isomerase and d-tagaturonate reductase represents an important step toward metabolic engineering of S. cerevisiae for bioethanol production from d-galacturonate. To investigate in vivo activity of the first steps of the d-galacturonate pathway, the L. lactis uxaB and uxaC genes were expressed in a gpd1¿ gpd2¿ S. cerevisiae strain. Although d-tagaturonate reductase could, in principle, provide an alternative means for re-oxidizing cytosolic NADH, addition of d-galacturonate did not restore anaerobic growth, possibly due to absence of a functional d-altronate exporter in S. cerevisiae.

Lactobacillus delbrueckii subsp . bulgaricus LBB.B5 originates from homemade Bulgarian yogurt and was selected for its ability to form a strong association with Streptococcus thermophilus . The genome sequence will facilitate elucidating the genetic background behind the contribution of LBB.B5 to the taste and aroma of yogurt and its exceptional protocooperation with S. thermophilus .

Iron is an important element for bacterial viability, however it is not readily available in most environments. We studied the ability of 20 undomesticated food isolates of Bacillus cereus and two reference strains for capacity to use different (complex) iron sources for growth and biofilm formation. Studies were performed in media containing the iron scavenger 2,2-Bipyridine. Transcriptome analysis using B. cereus ATCC 10987 indeed showed upregulation of predicted iron transporters in the presence of 2,2-Bipyridine, confirming that iron was depleted upon its addition. Next, the impact of iron sources on growth performance of the 22 strains was assessed and correlations between growth stimulation and presence of putative iron transporter systems in the genome sequences were analyzed. All 22 strains effectively used Fe citrate and FeCl3 for growth, and possessed genes for biosynthesis of the siderophore bacillibactin, whereas seven strains lacked genes for synthesis of petrobactin. Hemoglobin could be used by all strains with the exception of one strain that lacked functional petrobactin and IlsA systems. Hemin could be used by the majority of the tested strains (19 of 22). Notably, transferrin, ferritin, and lactoferrin were not commonly used by B. cereus for growth, as these iron sources could be used by 6, 3, and 2 strains, respectively. Furthermore, biofilm formation was found to be affected by the type of iron source used, including stimulation of biofilms at liquid-air interphase (FeCl3 and Fe citrate) and formation of submerged type biofilms (hemin and lactoferrin). Our results show strain variability in the genome-encoded repertoire of iron-transporting systems and differences in efficacy to use complex iron sources for growth and biofilm formation. These features may affect B. cereus survival and persistence in specific niches.

Blood groups, finger prints, iris scans and DNA bar codes are several ways to distinguish or identify individual humans. According to Arumugam and colleagues (2011) we can now also be distinguished by the microbial communities in our faeces, our ‘enterotypes’. The human gut is one of the most densely populated ecosystems known, and although this ecosystem contains members of the three domains of life – bacteria, archaea and eucarya (Finegold et al., 1983) – it is dominated by bacteria. They are essential in digesting the food we eat and in keeping us healthy by stimulating our immune system and fighting off pathogenic bacteria. It is estimated that 1013–1014 microbes inhabit our gastrointestinal tract (GIT), with the greatest number residing in the distal gut, where they synthesize essential vitamins and process otherwise indigestible components of our diet such as plant polysaccharides (Backhed et al., 2005). Early studies used 16S ribosomal DNA (rDNA) analysis to type and enumerate the distal gut and faecal microbiota. More than 90% of the bacterial phylotypes present in the intestinal microbiota in healthy humans are members of only three bacterial divisions: the Bacteroidetes, the Firmicutes and the Actinobacteria (Zoetendal et al., 2006). Nevertheless, each healthy adult's gut appears to have a unique and relatively stable microbiota (Zoetendal et al., 1998; Turnbaugh et al., 2007), which is a reflection of the numerous different phylogenetic clusters among the Firmicutes, Clostridium clusters IV, IX and XIVa, including the predominant genera Clostridium, Eubacterium, Roseburia and Ruminococcus. Furthermore, the Actinobacteria that encompass mainly the genera Bifidobacterium and Atopobium also represent important members of the gut microbial community (Harmsen et al., 2002; Turroni et al., 2008). Notably, recent estimates of the diversity of the human gut microbial ecosystem indicate it may encompass more than 1000 species and a multitude of strains (Backhed et al., 2005; Blaut and Clavel, 2007; Rajilic‐Stojanovic et al., 2007). Microbiota composition studies in humans have discovered that aberrations in the microbiome composition is present in obese individuals (Ley et al., 2006; Turnbaugh et al., 2009) as well as in individuals with a variety of other diseases (Zoetendal et al., 2008). Since the largest part (∼80%) of gut microbes remains uncultured, metagenomics analysis has become fashionable in the past 5 years to estimate the types, relative abundance and genome content of microbes in various parts of the GIT. The first metagenomics analysis from just two faecal samples (Gill et al., 2006) led to early insight into enrichment of genes encoding specific metabolic pathways, including metabolism associated with glycans, amino acids and xenobiotics, but also methanogenesis and biosynthesis of vitamins and isoprenoids. However, this early study provided only a very fragmented view due to limitations in sample size, sequencing technology, number of bases sequenced and availability of suitable reference genomes of gut inhabitants.

Microbial genomics and related transcriptomics methods rely on culturing techniques to obtain enough DNA suitable for high‐throughput sequencing without resorting to DNA amplification techniques. A few microgram of DNA is needed for most common next‐generation sequencing methods. For transcriptome analysis, sufficient cDNA is needed to measure low abundance mRNA copies in the cell. However, the large majority of microbes on earth resist cultivation, hampering research into their relevant gene pool, ecological niche or industrial relevance. For example, many environmental or gut‐related species cannot be grown outside their natural habitat. Even if we isolate the metagenome or the metatranscriptome from these environments, this reveals only a fragmented sequence landscape that is difficult to assign to individual species. Although enrichment techniques or metatransciptome analysis of previously unculturable species have been shown to assist in directed culturing, e.g. of a Rikenella‐like bacterium (Bomar et al., 2011), the unravelling of a complex metagenome into its individual genomes and their organization is impossible using current technologies. A major challenge is the analysis of bacteria and other organisms living inside a complex matrix, like biofilms. Metagenome or transcriptome analysis of microorganisms has been described for biofilms consisting of a single species by scraping of the biofilm to obtain enough material (Holmes et al., 2006), but for multi‐species biofilms this method results in a metagenome or metatranscriptome dataset. The solution to these challenges may be the isolation and genomic analysis of unculturable single cells isolated from such environments. Here we describe in brief the state‐of‐the‐art in single‐cell microbial genomics.

As the price of genome sequencing has been rapidly decreasing and can be expected to keep on doing so in the next 10 years, the speed at which new microbial genome sequences become available will increase accordingly. In most genome projects, the first step after acquiring a genome sequence is predicting protein‐encoding open reading frames (pORFs). Small proteins or peptides, loosely defined as less than 50 amino acids, encoded in microbial genomes have been largely underestimated. Recent focused functional genomics efforts have led to the identification of a number of new small proteins encoded in genomes of both Gram‐negative and Gram‐positive bacteria, and fungi (Kastenmayer et al., 2006; Li et al., 2008; Hemm et al., 2010; Hobbs et al., 2010; Bitton et al., 2011). Increasing evidence demonstrates that small proteins participate in a wide array of cellular processes and exhibit great diversity in their mechanisms of action. A recent review (Hobbs et al., 2011) highlights examples of small proteins that, in addition to the well‐conserved small ribosomal proteins, participate in cell signalling or regulation, act as antibiotics and toxins/anti‐toxins, alter membrane features, act as chaperones, stabilize protein complexes or serve as structural proteins (Table 1) (Fig. 1). Table 1 Types and functions of small proteins. Figure 1 Structure of small proteins. Small secreted proteins exhibit diversity in their three‐dimensional structures and can contain unique intramolecular linkages or modified amino acids. For example, the mature form of (A) subtilosin (PDB: 1PXQ) is ... Failure to recognize a pORF encoding a small protein means that these important cell constituents will be missed. Here, we give a brief summary of which problems arise in searching for such encoded small proteins, and what we could do to improve the search process.

Summary Lactococcus lactis produces lactic acid and is widely used in the manufacturing of various fermented dairy products. However, the species is also frequently isolated from non-dairy niches, such as fermented plant material. Recently, these non-dairy strains have gained increasing interest, as they have been described to possess flavour-forming activities that are rarely found in dairy isolates and have diverse metabolic properties. We performed an extensive whole-genome diversity analysis on 39 L. lactis strains, isolated from dairy and plant sources. Comparative genome hybridization analysis with multistrain microarrays was used to assess presence or absence of genes and gene clusters in these strains, relative to all L. lactis sequences in public databases, whereby chromosomal and plasmid-encoded genes were computationally analysed separately. Nearly 3900 chromosomal orthologous groups (chrOGs) were defined on basis of four sequenced chromosomes of L. lactis strains (IL1403, KF147, SK11, MG1363). Of these, 1268 chrOGs are present in at least 35 strains and represent the presently known core genome of L. lactis, and 72 chrOGs appear to be unique for L. lactis. Nearly 600 and 400 chrOGs were found to be specific for either the subspecies lactis or subspecies cremoris respectively. Strain variability was found in presence or absence of gene clusters related to growth on plant substrates, such as genes involved in the consumption of arabinose, xylan, a-galactosides and galacturonate. Further nichespecific differences were found in gene clusters for exopolysaccharides biosynthesis, stress response (iron transport, osmotolerance) and bacterial defence mechanisms (nisin biosynthesis). Strain variability of functions encoded on known plasmids included proteolysis, lactose fermentation, citrate uptake, metal ion resistance and exopolysaccharides biosynthesis. The present study supports the view of L. lactis as a species with a very flexible genome.

A novel 51-kb conjugative transposon of Lactococcus lactis , designated Tn 6098 , encoding the capacity to utilize α-galactosides such as raffinose and stachyose, was identified and characterized. Alpha-galactosides are a dominant carbon source in many plant-derived foods. Most dairy lactococcus strains are unable to use α-galactosides as a growth substrate, yet many of these strains are known to have beneficial industrial traits. Conjugal transfer of Tn 6098 was demonstrated from the plant-derived donor strain L. lactis KF147 to the recipient L. lactis NZ4501, a derivative of the dairy model strain L. lactis MG1363. The integration of Tn 6098 into the genome of the recipient strain was confirmed by Illumina sequencing of the transconjugant L. lactis NIZO3921. The molecular structure of the integration site was confirmed by a PCR product spanning the insertion site. A 15-bp direct repeat sequence (TTATACCATAATTAC) is present on either side of Tn 6098 in the chromosome of L. lactis KF147. One copy of this sequence is also present in the L. lactis MG1363 chromosome and represents the sole integration site. Phenotypic characterization of all strains showed that the transconjugant has not only acquired the ability to grow well in soy milk, a substrate rich in α-galactosides, but also has retained the flavor-forming capabilities of the recipient strain L. lactis MG1363. This study demonstrates how (induced) conjugation can be used to exploit the beneficial industrial traits of industrial dairy lactic acid bacteria in fermentation of plant-derived substrates.

The “Harlingen” IS 6110 restriction fragment length polymorphism (RFLP) cluster has linked over 100 tuberculosis cases in The Netherlands since 1993. Four Mycobacterium tuberculosis isolates that were epidemiologically linked to this cluster had different spoligotype patterns, as well as slightly divergent IS 6110 profiles, compared to the majority of the isolates. Sequencing of the direct repeat (DR) locus revealed sequence polymorphisms at the putative deletion sites. These deletion footprints provided evidence for independent deletions of the central region of the DR locus in three isolates, while the different genotype of the fourth isolate was explained by transmission. Our finding suggests that convergent deletions in the DR locus occur frequently. However, deletion footprints are not suitable to detect convergent deletions in the DR because they seem to be exceptional. Deletion footprints in the DR were not described previously, and we did not observe them in any public M. tuberculosis complex sequences. We conclude that preferential deletions in the DR loci of closely related strains are usually an unnoted event that interferes with clustering of closely related strains.

Summary Gene regulatory networks can be reconstructed by combining transcriptome data from many different experiments to elucidate relations between the activity of certain transcription factors and the genes they control. To obtain insight in the regulatory network of Lactobacillus plantarum, microarray transcriptome data from more than 70 different experimental conditions were combined and the expression profiles of the transcriptional units (TUs) were compared. The TUs that displayed correlated expression were used to identify putative cis-regulatory elements by searching the upstream regions of the TUs for conserved motifs. Predicted motifs were extended and refined by searching for motifs in the upstream regions of additional TUs with correlated expression. In this way, cis-acting elements were identified for 41 regulons consisting of at least four TUs (correlation > 0.7). This set of regulons included the known regulons of CtsR and LexA, but also several novel ones encompassing genes with coherent biological functions. Visualization of the regulons and their connections revealed a highly interconnected regulatory network. This network contains several subnetworks that encompass genes of correlated biological function, such as

Tracing the source of an infectious human disease can save lives. It allows for measures to be taken to prevent further spread of the disease. Although the mode of transmission for many human pathogens is known, it often remains difficult to trace the exact source of an outbreak of a disease with laboratory methods. Viruses, bacteria, fungi, parasites and protozoa can cause human diseases, but here we focus on bacterial pathogens. The currently used techniques to obtain DNA fingerprints of bacterial agents of infectious diseases frequently cannot discriminate between all bacterial strains of the same outbreak, making it impossible to follow the spread of the disease. A recent solution to this problem is the application of next‐generation whole‐genome sequencing techniques, which allows all available genetic information of each clinical isolate to be determined.

The genus Lactobacillus includes a diverse group of bacteria consisting of many species that are associated with fermentations of plants, meat or milk. In addition, various lactobacilli are natural inhabitants of the intestinal tract of humans and other animals. Finally, several Lactobacillus strains are marketed as probiotics as their consumption can confer a health benefit to host. Presently, 154 Lactobacillus species are known and a growing fraction of these are subject to draft genome sequencing. However, complete genome sequences are needed to provide a platform for detailed genomic comparisons. Therefore, we selected a total of 20 genomes of various Lactobacillus strains for which complete genomic sequences have been reported. These genomes had sizes varying from 1.8 to 3.3 Mb and other characteristic features, such as G+C content that ranged from 33% to 51%. The Lactobacillus pan genome was found to consist of approximately 14 000 protein-encoding genes while all 20 genomes shared a total of 383 sets of orthologous genes that defined the Lactobacillus core genome (LCG). Based on advanced phylogeny of the proteins encoded by this LCG, we grouped the 20 strains into three main groups and defined core group genes present in all genomes of a single group, signature group genes shared in all genomes of one group but absent in all other Lactobacillus genomes, and Group-specific ORFans present in core group genes of one group and absent in all other complete genomes. The latter are of specific value in defining the different groups of genomes. The study provides a platform for present individual comparisons as well as future analysis of new Lactobacillus genomes.

Rising sea levels, global pollution, economic meltdown and general prophecies of doom and gloom are inspired by the pending depletion of the world fossil carbon stores. With modern lifestyle on the rise, and an ever increasing world population driven on by economic advantage, we burn up earth's stores of mineralized corpses and vent CO2 as if there is no tomorrow. The billion dollar question for our society is: Where to get the next big, and prefer- ably clean, fuel injection? Fossil carbon compounds, the incompletely biodegraded remains of animals and plants long gone, originate from atmospheric CO2 via photosyn- thesis. Therefore, photosynthesis as main driver for gen- eration of de novo fuels has gained attention. First- and second-generation biofuels envision the conversion of plant biomass via the action of microorganisms, into usable organic compounds (alcohols and fats) and hydro- gen. However, water and land misuse, deforestation, and rising food prices, for the sake of growing choice biomass- producing crops, have raised major concerns (Frow et al., 2009; Young, 2009). Third-generation biofuels (and chemicals) produced with microalgae have now come forward as an answer to many of these concerns (Tredici, 2010). In general, microalgae do not comprise an evolu- tionarily related group, and may refer to cyanobacteria (blue-green algae) or eukaryotic algae. Even the eukary- otic algae do not form a single evolutionary branch as it can be used to indicate 'plant algae' such as red algae (Rhodophyta), brown algae (Phaetophyta) and green algae (Chlorophyta) or diatoms (Fig. 1). Some species of microalgae naturally accumulate vast quantities of oils, some above 80% dry-weight (Fig. 2). In comparison, agricultural oil-producing crops such as palm oil and soybean rarely produce more than 5% dry-weight in oils. Furthermore, algae can be grown everywhere where there is plenty of water and sun (including lakes or in the sea) and thus are not necessarily restricted to (or compete with) areas with arable land. Combined with their fast growth rate, microalgae are considered one of the few realistic sources for the production of biofuels and supe- rior to agricultural crop-derived bioethanol (Chisti, 2007; Tredici, 2010). Indeed, in 2009 ExxonMobil pledged $600 million for research in support of photosynthetic algae biofuels programmes, including Craig Venter's Synthetic Genomics. This signals a growing interest in de novo photosynthesis-derived fuels even by established oil- producing companies. Here we present a genomics update of some of Mother Nature's finest microorganisms which can make 'something from nothing' with a breath of CO2, a gulp of water, and all the while basking in the sun. Meet the cyanobacteria, green algae and (photosynthetic) diatoms that may lend a helping hand to fuel the human race.

These days, more and more scientists are diving into genome sequencing projects, urged by fast and cheap next‐generation sequencing technologies. Only to discover that they are quickly drowning in an unfathomable sea of sequence data and gasping for help from experts to make biological sense of this ensuing disaster. Bioinformaticians and genome annotators to the rescue! Microbial genome annotation involves primarily identifying the genes (or actually the open reading frames: ORFs) encrypted in the DNA sequence and deducing functionality of the encoded protein and RNA products (Fig. 1). First, a gene finder such as Glimmer (Delcher et al., 1999) or GeneMark (Lukashin and Borodovsky, 1998) is applied to the genome DNA sequence, producing a set of predicted protein‐coding genes. These programs are quite accurate, though not perfect. The next step is to take the set of predictions and search for hits against one or more protein and/or protein domain databases using blast (Altschul et al., 1997), HMMer (Eddy, 1998) or other programs. For each gene that has a significant match, the blast output together with the annotation of the hit can be used to assign a name and function to the protein. The accuracy of this step depends not only on the annotation software, but also on the quality of the annotations already in the reference database. Figure 1 A generalised flow chart of genome annotation. Statistical gene prediction: use of methods like GeneMark or Glimmer to predict protein‐coding genes. General database search: searching sequence databases (typically, NCBI NR) for sequence similarity, ... Genome sequences deposited in NCBI/GenBank, EMBL and DDBJ databases (which mirror each other) are annotated by the submitting groups, who each use their own methods, criteria and thoroughness. This leads to a large diversity in annotation completeness and accuracy. Many of the first genomes published had very limited or no functional annotation, simply because there was very little genomic information in these reference databases to compare with. Most public genome annotation remains static for years, and many annotations have never been changed since their initial publication. Over the years, annotation updates may have been maintained by the submitters, but they are generally only stored in local databases such as GenProtEC/EcoGene for Escherichia coli K12 (Rudd, 2000), Genolist/Bactilist for Bacillus subtilis 168 (Lechat et al., 2008) and SGD for Saccharomyces cerevisiae (Christie et al., 2004). Since gene functional annotation relies heavily on sequence similarity searching techniques with protein sequence databases, automatically annotated entries based on blast hits to NCBI databases can quickly become outdated. In the mean time, downstream sciences, such as comparative genomics, proteomics, transcriptomics and metabolomics, have rapidly increased our knowledge of many gene products. It is critical therefore, that genome annotations are frequently updated if the information they contain is to remain accurate, relevant and useful.

Hybridization to microarrays has been the standard for genome‐wide transcriptome analyses of prokaryotes in the past 10 years. Microarrays have several limitations, however, among which are a small dynamic range for detection of transcript levels due to problems with saturation, background noise, spot density and spot quality. Moreover, comparing different experiments requires complex normalization methods (Hinton et al., 2004) and comparing different strains requires designing pangenome arrays based on multiple sequenced genomes, leading to further problems in non‐specific or cross‐hybridization and complicated data analysis (Bayjanov et al., 2009). Most microarrays have a biased genome coverage, as they only contain a limited number of short probes for known or expected genes in sequenced genomes, and they rarely probe intergenic regions. Technological advances in array production and dropping costs have recently led to the design and use of high‐density tiling arrays based on overlapping short oligonucleotides covering both strands of entire genomes (Selinger et al., 2000; Mcgrath et al., 2007; Rasmussen et al., 2009; Toledo‐Arana et al., 2009). Tiling array and other studies have provided a first insight into far more complex transcriptomes than previously envisioned, including an ever‐expanding range of regulatory RNAs (Waters and Storz, 2009). To overcome the remaining limitations of microarrays, a totally new approach to whole‐transcriptome analysis was needed – and a much‐awaited breakthrough in DNA sequencing came to the rescue. Here, we describe the first whole‐transcriptome applications in prokaryotes and discover that a new treasure chest of regulation in prokaryotes is being opened.

Contains fulltext : 87731.pdf (Publisher’s version ) (Closed access) A total of six polymorphisms were identified by comparing the genomes of the first and the last isolate of a well-characterized transmission chain of Mycobacterium tuberculosis involving five patients over a 12 and a half year period. The six polymorphisms consisted of four single nucleotide changes (SNPs), a tandem repeat polymorphism (TRP) and a previously identified IS6110 transposition event. These polymorphic sites were surveyed in each of the isolates from the five patients in the transmission chain. Surprisingly, five of the six polymorphisms accumulated in a single patient in the transmission chain; this patient had been non-compliant to tuberculosis treatment. This first insight into the tempo and mode of molecular evolution in M. tuberculosis at the patient-to-patient level suggests that the molecular evolution of the pathogen in vivo is characterized by periods of relative genomic stability followed by bursts of mutation. Whatever the mechanism for the accumulation of mutations, this observation may have profound consequences for the application of vaccines and therapeutic drugs, the management and treatment of disease outbreaks of M. tuberculosis, the most important bacterial pathogen of humans. 01 januari 2010

Crude oil (petroleum) is a highly complex mixture of organic compounds of which some 1.3 million litres enters the environment each year. More then anything else, the numerous oil‐shipping disasters, such as of the Exxon Valdez (1989), the Erika (1999) and the Prestige (2003), have captured the public attention to this environmental problem (Fig. 1). However, these accidents account for only a small part of the annual global release of crude oil, as most enters the environment from deliberate discharge and processing sites. Around three million tons of oil enters the sea each year, of which about 20% originates from oil‐pumping operations, transport and refining activities and 25% from non‐tanker shipping and natural seepages. More than half (55%) originates from illegal activities that include the dumping of ballast water and oil residues as well as accidents (Golyshin et al., 2003). Hydrocarbons are also produced continuously by living cells as natural oils and fats (de Lorenzo, 2006). The observation that the oceans are not covered with an oily layer is a testimony to the activity of the hydrocarbon‐degrading microorganisms (Head et al., 2006). Several bacteria are even known to feed exclusively on hydrocarbons (Yakimov et al., 2007). For these (facultative) hydrocarbon degraders the occasional supertanker oil spill forms an occasional carbon banquet. They play an important role in the clean‐up after an oil spill and form the biological basis for the natural oil‐degrading capacity of the ecosystem. Studies have focused on identifying and characterizing these oil‐eating microbes, as well as how they cope with the oil/water interface, and how to improve this capacity. Here we highlight some of the recent genomics advances in this field.

Plant-associated microbes and plant–microbe interactions can be largely divided in two types: detrimental (pathogenic) and beneficial (symbiotic) interactions. Neutral interactions also occur; this is the case for microbes that live in the rhizosphere (on roots) or phyllosphere (on leaves) without triggering any apparent plant response. Both pathogenic and symbiotic interactions are of relevance to industry because these may impact plant production in negative and positive ways respectively. Several plant-associated microbes produce cell wall-degrading enzymes which may be of industrial use in fermentation of plant products. Here we give a brief update of the current status of genome sequencing and genomics of microbes associated with plants.

Over two‐thirds of the surface of the earth is covered by oceans, which have an average depth of about 3800 m. As each drop of ocean water contains > 105 cells, the > 1030 microbial cells in the ocean represent the largest reservoir of microbes on earth (Whitman et al., 1998). Communities of bacteria, archaea, protists and unicellular fungi account for most of the oceanic biomass and metabolism. Marine microbes are known to play an essential role in the global cycling of nitrogen, carbon, oxygen, phosphorous, iron, sulfur and trace elements (Karl, 2007).

Motivation: Pangenome arrays contain DNA oligomers targeting several sequenced reference genomes from the same species. In microbiology, these can be employed to investigate the often high genetic variability within a species by comparative genome hybridization (CGH). The biological interpretation of pangenome CGH data depends on the ability to compare strains at a functional level, particularly by comparing the presence or absence of orthologous genes. Due to the high genetic variability, available genotype-calling algorithms can not be applied to pangenome CGH data. Results: We have developed the algorithm PanCGH that incorporates orthology information about genes to predict the presence or absence of orthologous genes in a query organism using CGH arrays that target the genomes of sequenced representatives of a group of microorganisms. PanCGH was tested and applied in the analysis of genetic diversity among 39 Lactococcus lactis strains from three different subspecies (lactis.cremoris, hordniae) and isolated from two different niches (dairy and plant). Clustering of these strains using the presence/absence data of gene orthologs revealed a clear separation between different subspecies and reflected the niche of the strains. Contact: J.Bayjanov@cmbi.ru.nl Supplementary information: Supplementary data are available at Bioinformatics online.

Cleaning up wastewater from domestic sewage and industrial processes is essential for safeguarding public health and the environment (for an overview of wastewater treatment processes see http://en.wikipedia.org/wiki/Sewage_treatment). Biological wastewater treatment plants (WWTPs) employing sludge represent one of the most widely used biotechnological processes, with more than 120 billion litres treated daily in the USA alone (Fig. 1). Removal of organic carbon and other nutrients, mainly nitrogen (N) and phosphorus (P), by sludge microbes is essential in order to avoid eutrophication and deterioration of recipient surface waters (Seviour et al., 2003). The complexity of wastewater microbial communities, based on the analysis of 16S rRNA sequences, is known to be enormous (Chouari et al., 2005a,b) (Fig. 2). As we have very limited knowledge of composition, dynamics and stability of microbial communities, several processes in wastewater treatment are generally regarded as being a ‘black box’. Their improvement is often based on a trial‐and‐error approach, which leads to inconsistent results and unexpected behaviours (Daims et al., 2006a). In recent years, with the development of several new high‐throughput sequencing platforms (Hall, 2007; Marsh, 2007), metagenome sequencing strategies (Schloss and Handelsman, 2005; Tringe and Rubin, 2005; Schmeisser et al., 2007) and bioinformatics toolboxes (Chen and Pachter, 2005; Tringe et al., 2005; Foerstner et al., 2006; Raes et al., 2007), the genome analysis of complex communities has become much more accessible, albeit by no means easier. Here we give a brief update of the current status of (meta)genome sequencing and mining of wastewater‐related microorganisms and communities. Figure 1 Wastewater treatment plant near Arnhem, the Netherlands (photo M. Galardini). Figure 2 Bacterial diversity analysis in an anaerobic sludge digester. Neighbour‐joining tree of bacterial 16S rRNA sequences showing different operational taxonomic units (OTUs). Number of OTUs is indicated on the branches, and total number of clones ...

Noroviruses (NoVs) are considered to be a major cause of acute nonbacterial gastroenteritis in humans. The NoV genus is genetically diverse, and genotype GII.4 has been most commonly identified worldwide in recent years. In this study we analyzed the complete capsid gene of NoV strains belonging to the less prevalent genotype GII.2. We compared a total of 36 complete capsid sequences of GII.2 sequences obtained from the GenBank ( n = 5) and from outbreaks or sporadic cases that occurred in The Netherlands ( n = 10) and in Osaka City, Japan ( n = 21), between 1976 and 2005. Alignment of all capsid sequences did not show fixation of amino acid substitutions over time as an indication for genetic drift. In contrast, when strains previously recognized as recombinants were excluded from the alignment, genetic drift was observed. Substitutions were found at five informative sites (two in the P1 subdomain and three in the P2 subdomain), segregating strains into five genetic groups (1994 to 1997, 1999 to 2000, 2001 to 2003, 2004, and 2005). Only one amino acid position changed consistently between each group (position 345). Homology modeling of the GII.2 capsid protein showed that the five amino acids were located on the surface of the capsid and close to each other at the interface of two monomers. The data suggest that these changes were induced by selective pressure, driving virus evolution. Remarkably, this was observed only for nonrecombinant genomes, suggesting differences in behavior with recombinant strains.

In bacteria, environmental challenges are often translated into a transcriptional response via the cognate response regulators (RRs) of specialized two-component systems (TCSs). A phylogenetic footprinting/shadowing approach was designed and used to identify many novel RR-specific operators for species of the Bacillus cereus group and related Gram-positives. Analysis of the operator sequences revealed characteristic traits for each RR subfamily. For instance, operators related to the largest subfamily (OmpR) typically consisted of direct repeats (e.g. TTAAGA-N5-TTAAGA), whereas operators related to the second largest family (NarL) consisted of inverted repeats (e.g. ATGACA-N2-TGTCAT). This difference indicates a fundamentally different organization of the bound RR dimers between the two subfamilies. Moreover, the identification of the specific operator motifs allowed relating several RRs to a minimal regulon and thereby to a characteristic transcriptional response. Mostly, these regulons comprised genes encoding transport systems, suggesting a direct coupling of stimulus perception to the transport of target compounds. New biological roles could be attributed to various TCSs, including roles in cytochrome c biogenesis (HssRS), transport of carbohydrates, peptides and/or amino acids (YkoGH, LytSR), and resistance to toxic ions (LiaSR), antimicrobial peptides (BceRS) and beta-lactam antibiotics (BacRS, YcbLM). As more and more bacterial genome sequences are becoming available, the use of comparative analyses such as the approach applied in this study will further increase our knowledge of bacterial signal transduction mechanisms and provide directions for the assessment of their role in bacterial performance and survival strategies.

Secondary metabolites (or natural products) are often synthesized by multi‐modular, multi‐domain proteins called non‐ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS). Various well‐known metabolites produced by microorganisms are listed in Table 1, and examples of structures are shown in Fig. 1. In particular, Streptomyces species are known for their ability to produce a wide variety of secondary metabolites such as antibiotics, herbicides, parasitocides, siderophores and pharmacologically active substances including antitumour agents and immunosuppressants. Genome sequencing of Streptomyces coelicolor (Bentley et al., 2002) and S. avermitilis (Omura et al., 2001) revealed over 20 gene clusters for biosynthesis of secondary metabolites, while only a few of their natural products were known prior to sequencing. High‐throughput genome sequencing of hundreds of other bacterial species and strains is now rapidly increasing the repertoire of identified gene clusters for biosynthesis of natural products (Donadio et al., 2007). Here we give a brief update of the current status of genome mining and bioinformatic tools to identify novel NRPS and PKS systems. Table 1 Examples of microbial natural products produced by NRPS/PKS systems. Figure 1 Examples of some chemical structures of (A) polyketides, (B) non‐ribosomal peptides and (C) mixed NRP‐PK compounds. Reprinted with permission from Watanabe and Oikawa (2007). Copyright Royal Society of Chemistry.

In the past few years, the number of microbial genome sequencing projects worldwide has rapidly increased, both of single species and microbial consortia (metagenomes). The development of several new high‐throughput sequencing platforms (Hall, 2007; Marsh, 2007), and an enormous reduction in costs, means we can expect to have thousands of complete and incomplete genomes sequences available to us in the coming years. Many of these microbial genomes are of biotechnological interest, and several have spectacular properties in relation to their growth requirements, the metabolites they produce, their potential for environmental clean‐up or survival in extreme environments. One of the ideas behind sequencing and analysis of whole genomes or substantial parts is that it will be used to enable a more targeted construction of mutant strains for improvement of industrial processes. This is in contrast to the more common procedure of production of random mutations and then screening for the desired phenotype. The sheer number of newly completed genomes, estimated at about 1 per day in 2008, makes it impossible to publish all this information in regular scientific journals. So how do we keep track of which genome sequences are known or are upcoming, and where can we find all this sequence data to do data mining and comparative genomics in search of leads for our own research on biotechnologically interesting microbes?

Contains fulltext : 69805.pdf (Publisher’s version ) (Closed access) Most Gram-positive bacteria inhabiting the gastrointestinal tract are capable of hydrolysing bile salts. Bile salt hydrolysis is thought to play an important role in various biological processes in the host. Therefore, correct annotation of bacterial bile salt hydrolases (Bsh) in public databases (EC 3.5.1.24) is of importance, especially for lactobacilli, which are considered to play a major role in bile salt hydrolysis in vivo. In the present study, all enzymes listed in public databases that belong to the Bsh family and the closely related penicillin V acylase (Pva; EC 3.5.1.11) family were compared with the sequences annotated as Bsh in Lactobacillus plantarum WCFS1, as an example. In Gram-positive bacteria, a clear distinction was made between the two families using sequence alignment, phylogenetic clustering, and protein homology modelling. Biochemical and structural data on experimentally verified Bsh and Pva enzymes were used for validation of function prediction. Hidden Markov models were constructed from the sequence alignments to enable a more accurate prediction of Bsh-encoding genes, and their distinction from those encoding members of the Pva family. Many Pva-related sequences appeared to be annotated incorrectly as Bsh in public databases. This refinement in the annotation of Bsh family members influences the prediction of the function of bsh-like genes in species of the genus Lactobacillus, and it is discussed in detail.

Item does not contain fulltext Mucosal pentraxin (Mptx), identified in rats, is a short pentraxin of unknown function. Other subfamily members are Serum amyloid P component (SAP), C-reactive protein (CRP) and Jeltraxin. Rat Mptx mRNA is predominantly expressed in colon and in vivo is strongly (30-fold) regulated by dietary heme and calcium, modulators of colon cancer risk. This renders Mptx a potential nutrient sensitive biomarker of gut health. To support a role as biomarker, we examined whether the pentraxin protein structure is conserved, whether Mptx protein is nutrient-sensitively expressed and whether Mptx is expressed in mouse and human. Sequence comparison and 3D modelling showed that rat Mptx is highly homologous to the other pentraxins. The calcium-binding site and subunit interaction sites are highly conserved, while a loop deletion and charged residues contribute to a distinctive �top� face of the pentamer. In accordance with mRNA expression, Mptx protein is strongly down-regulated in rat colon mucosa in response to high dietary heme intake. Mptx mRNA is expressed in rat and mouse colon, but not in human colon. A stop codon at the beginning of human exon two indicates loss of function, which may be related to differences in intestinal cell turnover between man and rodents.

Contains fulltext : 34793renckens.pdf (Publisher’s version ) (Open Access) Noroviruses are the causative agents of the majority of viral gastroenteritis outbreaks in humans. During the past 15 years, noroviruses of genotype GGII.4 have caused four epidemic seasons of viral gastroenteritis, during which four novel variants (termed epidemic variants) emerged and displaced the resident viruses. In order to understand the mechanisms and biological advantages of these epidemic variants, we studied the genetic changes in the capsid proteins of GGII.4 strains over this period. A representative sample was drawn from 574 GGII.4 outbreak strains collected over 15 years of systematic surveillance in The Netherlands, and capsid genes were sequenced for a total of 26 strains. The three-dimensional structure was predicted by homology modeling, using the Norwalk virus (Hu/NoV/GGI.1/Norwalk/1968/US) capsid as a reference. The highly significant preferential accumulation and fixation of mutations (nucleotide and amino acid) in the protruding part of the capsid protein provided strong evidence for the occurrence of genetic drift and selection. Although subsequent new epidemic variants differed by up to 25 amino acid mutations, consistent changes were observed in only five positions. Phylogenetic analyses showed that each variant descended from its chronologic predecessor, with the exception of the 2006b variant, which is more closely related to the 2002 variant than to the 2004 variant. The consistent association between the observed genetic findings and changes in epidemiology leads to the conclusion that population immunity plays a role in the epochal evolution of GGII.4 norovirus strains.

Lactobacillus plantarum strain WJL is a symbiont isolated from the Drosophila melanogaster gut. The genome of L. plantarum WJL, first sequenced in 2013, was resequenced and rescaffolded in this study. A combination of Sanger and Illumina sequencing allowed us to reduce the number of contigs from 102 to 13. This work contributes to a better understanding of the genome and function of this organism.

Summary: We have developed CiVi, a user-friendly web-based tool to create custom circular maps to aid the analysis of microbial genomes and sequence elements. Sequence related data such as gene-name, COG class, PFAM domain, GC%, and subcellular location can be comprehensively viewed. Quantitative gene-related data (e.g. expression ratios or read counts) as well as predicted sequence elements (e.g. regulatory sequences) can be uploaded and visualized. CiVi accommodates the analysis of genomic elements by allowing a visual interpretation in the context of: (i) their genome-wide distribution, (ii) provided experimental data and (iii) the local orientation and location with respect to neighboring genes. CiVi thus enables both experts and non-experts to conveniently integrate public genome data with the results of genome analyses in circular genome maps suitable for publication. Contact: L.Overmars@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. Availability and implementation: CiVi is freely available at http://civi.cmbi.ru.nl

The lactic acid bacterium Streptococcus thermophilus is widely used for the fermentation of dairy products. Here, we present the draft genome sequence of S. thermophilus C106 isolated from an artisanal cheese produced in the countryside of Ireland.

Lactobacillus plantarum is a versatile bacterial species that is isolated mostly from foods. Here, we present the first genome sequence of L. plantarum strain NIZO2877 isolated from a hot dog in Vietnam. Its two contigs represent a nearly complete genome sequence.