The metagenome-derived esterase PET40 is highly promiscuous and hydrolyses polyethylene terephthalate (PET).

Polyethylene terephthalate (PET) is a widely used synthetic polymer and known to contaminate marine and terrestrial ecosystems. Only few PET-active microorganisms and enzymes (PETases) are currently known, and it is debated whether degradation activity for PET originates from promiscuous enzymes with broad substrate spectra that primarily act on natural polymers or other bulky substrates, or whether microorganisms evolved their genetic makeup to accepting PET as a carbon source. Here, we present a predicted diene lactone hydrolase designated PET40, which acts on a broad spectrum of substrates, including PET. It is the first esterase with activity on PET from a GC-rich Gram-positive Amycolatopsis species belonging to the Pseudonocardiaceae (Actinobacteria). It is highly conserved within the genera Amycolatopsis and Streptomyces. PET40 was identified by sequence-based metagenome search using a PETase-specific hidden Markov model. Besides acting on PET, PET40 has a versatile substrate spectrum, hydrolyzing δ-lactones, ß-lactam antibiotics, the polyester-polyurethane Impranil® DLN, and various para-nitrophenyl ester substrates. Molecular docking suggests that the PET degradative activity is likely a result of the promiscuity of PET40, as potential binding modes were found for substrates encompassing mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, and a PET trimer. We also solved the crystal structure of the inactive PET40 variant S178A to 1.60 Å resolution. PET40 is active throughout a wide pH (pH 4-10) and temperature range (4-65 °C) and remarkably stable in the presence of 5% SDS, making it a promising enzyme as a starting point for further investigations and optimization approaches.

The Bacteroidetes Aequorivita sp. and Kaistella jeonii Produce Promiscuous Esterases With PET-Hydrolyzing Activity.

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 µl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.

New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes.

Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and ß-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria, Proteobacteria, and Bacteroidetes Within the Proteobacteria, the Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria, as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil.IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET hydrolases. Finally, we describe the biochemical traits of four novel PET hydrolases.

The Absence of the N-acyl-homoserine-lactone Autoinducer Synthase Genes traI and ngrI Increases the Copy Number of the Symbiotic Plasmid in Sinorhizobium fredii NGR234.

Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be essential for initiating the plant symbiosis with rhizobia affiliated with the Alphaproteobacteria. Here, we provide evidence that in the broad host range strain Sinorhizobium fredii NGR234 the complete lack of quorum sensing molecules results in an elevated copy number of its symbiotic plasmid (pNGR234a). This in turn triggers the expression of symbiotic genes and the production of Nod factors in the absence of plant signals. Therefore, increasing the copy number of specific plasmids could be a widespread mechanism of specialized bacterial populations to bridge gaps in signaling cascades.

Phenotypic Heterogeneity Affects Stenotrophomonas maltophilia K279a Colony Morphotypes and ß-Lactamase Expression.

Phenotypic heterogeneity at the cellular level in response to various stresses, e.g., antibiotic treatment has been reported for a number of bacteria. In a clonal population, cell-to-cell variation may result in phenotypic heterogeneity that is a mechanism to survive changing environments including antibiotic therapy. Stenotrophomonas maltophilia has been frequently isolated from cystic fibrosis patients, can cause numerous infections in other organs and tissues, and is difficult to treat due to antibiotic resistances. S. maltophilia K279a produces the L1 and L2 ß-lactamases in response to ß-lactam treatment. Here we report that the patient isolate S. maltophilia K279a diverges into cellular subpopulations with distinct but reversible morphotypes of small and big colonies when challenged with ampicillin. This observation is consistent with the formation of elongated chains of bacteria during exponential growth phase and the occurrence of mainly rod-shaped cells in liquid media. RNA-seq analysis of small versus big colonies revealed differential regulation of at least seven genes among the colony morphotypes. Among those, bla L1 and bla L2 were transcriptionally the most strongly upregulated genes. Promoter fusions of bla L1 and bla L2 genes indicated that expression of both genes is also subject to high levels of phenotypic heterogeneous expression on a single cell level. Additionally, the comE homolog was found to be differentially expressed in homogenously versus heterogeneously bla L2 expressing cells as identified by RNA-seq analysis. Overexpression of comE in S. maltophilia K279a reduced the level of cells that were in a bla L2-ON mode to 1% or lower. Taken together, our data provide strong evidence that S. maltophilia K279a populations develop phenotypic heterogeneity in an ampicillin challenged model. This cellular variability is triggered by regulation networks including bla L1, bla L2, and comE.

Genome-wide RNA sequencing analysis of quorum sensing-controlled regulons in the plant-associated Burkholderia glumae PG1 strain.

Burkholderia glumae PG1 is a soil-associated motile plant-pathogenic bacterium possessing a cell density-dependent regulation system called quorum sensing (QS). Its genome contains three genes, here designated bgaI1 to bgaI3, encoding distinct autoinducer-1 (AI-1) synthases, which are capable of synthesizing QS signaling molecules. Here, we report on the construction of B. glumae PG1 ΔbgaI1, ΔbgaI2, and ΔbgaI3 mutants, their phenotypic characterization, and genome-wide transcriptome analysis using RNA sequencing (RNA-seq) technology. Knockout of each of these bgaI genes resulted in strongly decreased motility, reduced extracellular lipase activity, a reduced ability to cause plant tissue maceration, and decreased pathogenicity. RNA-seq analysis of all three B. glumae PG1 AI-1 synthase mutants performed in the transition from exponential to stationary growth phase revealed differential expression of a significant number of predicted genes. In comparison with the levels of gene expression by wild-type strain B. glumae PG1, 481 genes were differentially expressed in the ΔbgaI1 mutant, 213 were differentially expressed in the ΔbgaI2 mutant, and 367 were differentially expressed in the ΔbgaI3 mutant. Interestingly, only a minor set of 78 genes was coregulated in all three mutants. The majority of the QS-regulated genes were linked to metabolic activities, and the most pronounced regulation was observed for genes involved in rhamnolipid and Flp pilus biosynthesis and the type VI secretion system and genes linked to a clustered regularly interspaced short palindromic repeat (CRISPR)-cas gene cluster.

A comparative metagenome survey of the fecal microbiota of a breast- and a plant-fed Asian elephant reveals an unexpectedly high diversity of glycoside hydrolase family enzymes.

A phylogenetic and metagenomic study of elephant feces samples (derived from a three-weeks-old and a six-years-old Asian elephant) was conducted in order to describe the microbiota inhabiting this large land-living animal. The microbial diversity was examined via 16S rRNA gene analysis. We generated more than 44,000 GS-FLX+454 reads for each animal. For the baby elephant, 380 operational taxonomic units (OTUs) were identified at 97% sequence identity level; in the six-years-old animal, close to 3,000 OTUs were identified, suggesting high microbial diversity in the older animal. In both animals most OTUs belonged to Bacteroidetes and Firmicutes. Additionally, for the baby elephant a high number of Proteobacteria was detected. A metagenomic sequencing approach using Illumina technology resulted in the generation of 1.1 Gbp assembled DNA in contigs with a maximum size of 0.6 Mbp. A KEGG pathway analysis suggested high metabolic diversity regarding the use of polymers and aromatic and non-aromatic compounds. In line with the high phylogenetic diversity, a surprising and not previously described biodiversity of glycoside hydrolase (GH) genes was found. Enzymes of 84 GH families were detected. Polysaccharide utilization loci (PULs), which are found in Bacteroidetes, were highly abundant in the dataset; some of these comprised cellulase genes. Furthermore the highest coverage for GH5 and GH9 family enzymes was detected for Bacteroidetes, suggesting that bacteria of this phylum are mainly responsible for the degradation of cellulose in the Asian elephant. Altogether, this study delivers insight into the biomass conversion by one of the largest plant-fed and land-living animals.

RNA sequencing analysis of the broad-host-range strain Sinorhizobium fredii NGR234 identifies a large set of genes linked to quorum sensing-dependent regulation in the background of a traI and ngrI deletion mutant.

The alphaproteobacterium Sinorhizobium fredii NGR234 has an exceptionally wide host range, as it forms nitrogen-fixing nodules with more legumes than any other known microsymbiont. Within its 6.9-Mbp genome, it encodes two N-acyl-homoserine-lactone synthase genes (i.e., traI and ngrI) involved in the biosynthesis of two distinct autoinducer I-type molecules. Here, we report on the construction of an NGR234-ΔtraI and an NGR234-ΔngrI mutant and their genome-wide transcriptome analysis. A high-resolution RNA sequencing (RNA-seq) analysis of early-stationary-phase cultures in the NGR234-ΔtraI background suggested that up to 316 genes were differentially expressed in the NGR234-ΔtraI mutant versus the parent strain. Similarly, in the background of NGR234-ΔngrI 466 differentially regulated genes were identified. Accordingly, a common set of 186 genes was regulated by the TraI/R and NgrI/R regulon. Coregulated genes included 42 flagellar biosynthesis genes and 22 genes linked to exopolysaccharide (EPS) biosynthesis. Among the genes and open reading frames (ORFs) that were differentially regulated in NGR234-ΔtraI were those linked to replication of the pNGR234a symbiotic plasmid and cytochrome c oxidases. Biotin and pyrroloquinoline quinone biosynthesis genes were differentially expressed in the NGR234-ΔngrI mutant as well as the entire cluster of 21 genes linked to assembly of the NGR234 type III secretion system (T3SS-II). Further, we also discovered that genes responsible for rhizopine catabolism in NGR234 were strongly repressed in the presence of high levels of N-acyl-homoserine-lactones. Together with nodulation assays, the RNA-seq-based findings suggested that quorum sensing (QS)-dependent gene regulation appears to be of higher relevance during nonsymbiotic growth rather than for life within root nodules.

Evidence of autoinducer-dependent and -independent heterogeneous gene expression in Sinorhizobium fredii NGR234.

Populations of genetically identical Sinorhizobium fredii NGR234 cells differ significantly in their expression profiles of autoinducer (AI)-dependent and AI-independent genes. Promoter fusions of the NGR234 AI synthase genes traI and ngrI showed high levels of phenotypic heterogeneity during growth in TY medium on a single-cell level. However, adding very high concentrations of N-(3-oxooctanoyl-)-l-homoserine lactone resulted in a more homogeneous expression profile. Similarly, the lack of internally synthesized AIs in the background of the NGR234-ΔtraI or the NGR234-ΔngrI mutant resulted in a highly homogenous expression of the corresponding promoter fusions in the population. Expression studies with reporter fusions of the promoter regions of the quorum-quenching genes dlhR and qsdR1 and the type IV pilus gene cluster located on pNGR234b suggested that factors other than AI molecules affect NGR234 phenotypic heterogeneity. Further studies with root exudates and developing Arabidopsis thaliana seedlings provide the first evidence that plant root exudates have strong effects on the heterogeneity of AI synthase and quorum-quenching genes in NGR234. Therefore, plant-released octopine appears to play a key role in modulation of heterogeneous gene expression.

Metagenome survey of a multispecies and alga-associated biofilm revealed key elements of bacterial-algal interactions in photobioreactors.

Photobioreactors (PBRs) are very attractive for sunlight-driven production of biofuels and capturing of anthropogenic CO2. One major problem associated with PBRs however, is that the bacteria usually associated with microalgae in nonaxenic cultures can lead to biofouling and thereby affect algal productivity. Here, we report on a phylogenetic, metagenome, and functional analysis of a mixed-species bacterial biofilm associated with the microalgae Chlorella vulgaris and Scenedesmus obliquus in a PBR. The biofilm diversity and population dynamics were examined through 16S rRNA phylogeny. Overall, the diversity was rather limited, with approximately 30 bacterial species associated with the algae. The majority of the observed microorganisms were affiliated with Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes. A combined approach of sequencing via GS FLX Titanium from Roche and HiSeq 2000 from Illumina resulted in the overall production of 350 Mbp of sequenced DNA, 165 Mbp of which was assembled in larger contigs with a maximum size of 0.2 Mbp. A KEGG pathway analysis suggested high metabolic diversity with respect to the use of polymers and aromatic and nonaromatic compounds. Genes associated with the biosynthesis of essential B vitamins were highly redundant and functional. Moreover, a relatively high number of predicted and functional lipase and esterase genes indicated that the alga-associated bacteria are possibly a major sink for lipids and fatty acids produced by the microalgae. This is the first metagenome study of microalga- and PBR-associated biofilm bacteria, and it gives new clues for improved biofuel production in PBRs.

The Janthinobacterium sp. HH01 genome encodes a homologue of the V. cholerae CqsA and L. pneumophila LqsA autoinducer synthases.

Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes.

The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases.

Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75 °C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70 °C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70 °C. LipS had an optimum temperature at 70 °C and LipT at 75 °C. Both enzymes catalyzed hydrolysis of long-chain (C(12) and C(14)) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70 °C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.

The genome of the ammonia-oxidizing Candidatus Nitrososphaera gargensis: insights into metabolic versatility and environmental adaptations.

The cohort of the ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota is a diverse, widespread and functionally important group of microorganisms in many ecosystems. However, our understanding of their biology is still very rudimentary in part because all available genome sequences of this phylum are from members of the Nitrosopumilus cluster. Here we report on the complete genome sequence of Candidatus Nitrososphaera gargensis obtained from an enrichment culture, representing a different evolutionary lineage of AOA frequently found in high numbers in many terrestrial environments. With its 2.83 Mb the genome is much larger than that of other AOA. The presence of a high number of (active) IS elements/transposases, genomic islands, gene duplications and a complete CRISPR/Cas defence system testifies to its dynamic evolution consistent with low degree of synteny with other thaumarchaeal genomes. As expected, the repertoire of conserved enzymes proposed to be required for archaeal ammonia oxidation is encoded by N. gargensis, but it can also use urea and possibly cyanate as alternative ammonia sources. Furthermore, its carbon metabolism is more flexible at the central pyruvate switch point, encompasses the ability to take up small organic compounds and might even include an oxidative pentose phosphate pathway. Furthermore, we show that thaumarchaeota produce cofactor F420 as well as polyhydroxyalkanoates. Lateral gene transfer from bacteria and euryarchaeota has contributed to the metabolic versatility of N. gargensis. This organisms is well adapted to its niche in a heavy metal-containing thermal spring by encoding a multitude of heavy metal resistance genes, chaperones and mannosylglycerate as compatible solute and has the genetic ability to respond to environmental changes by signal transduction via a large number of two-component systems, by chemotaxis and flagella-mediated motility and possibly even by gas vacuole formation. These findings extend our understanding of thaumarchaeal evolution and physiology and offer many testable hypotheses for future experimental research on these nitrifiers.

Complete genome sequence of the broad-host-range strain Sinorhizobium fredii USDA257.

Here we announce the complete genome sequence of the symbiotic and nitrogen-fixing bacterium Sinorhizobium fredii USDA257. The genome shares a high degree of sequence similarity with the closely related broad-host-range strains S. fredii NGR234 and HH103. Most strikingly, the USDA257 genome encodes a wealth of secretory systems.

Rhizobium sp. strain NGR234 possesses a remarkable number of secretion systems.

Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. We report here that the 3.93-Mbp chromosome (cNGR234) encodes most functions required for cellular growth. Few essential functions are encoded on the 2.43-Mbp megaplasmid (pNGR234b), and none are present on the second 0.54-Mbp symbiotic plasmid (pNGR234a). Among many striking features, the 6.9-Mbp genome encodes more different secretion systems than any other known rhizobia and probably most known bacteria. Altogether, 132 genes and proteins are linked to secretory processes. Secretion systems identified include general and export pathways, a twin arginine translocase secretion system, six type I transporter genes, one functional and one putative type III system, three type IV attachment systems, and two putative type IV conjugation pili. Type V and VI transporters were not identified, however. NGR234 also carries genes and regulatory networks linked to the metabolism of a wide range of aromatic and nonaromatic compounds. In this way, NGR234 can quickly adapt to changing environmental stimuli in soils, rhizospheres, and plants. Finally, NGR234 carries at least six loci linked to the quenching of quorum-sensing signals, as well as one gene (ngrI) that possibly encodes a novel type of autoinducer I molecule.

Metagenomics, biotechnology with non-culturable microbes.

Metagenomics as a new field of research has been developed over the past decade to elucidate the genomes of the non-cultured microbes with the goal to better understand global microbial ecology on the one side, and on the other side it has been driven by the increasing biotechnological demands for novel enzymes and biomolecules. Since it is well accepted that the majority of all microbes has not yet been cultured, the not-yet-cultivated microbes represent a shear unlimited and intriguing resource for the development of novel genes, enzymes and chemical compounds for use in biotechnology. However, with respect to biotechnology, metagenomics faces now two major challenges. Firstly, it has to identify truly novel biocatalysts to fulfil the needs of industrial processes and green chemistry. Secondly, the already available genes and enzymes need to be implemented in production processes to further prove the value of metagenome-derived sequences.

Structural characterization of a K-antigen capsular polysaccharide essential for normal symbiotic infection in Rhizobium sp. NGR234: deletion of the rkpMNO locus prevents synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-non-2-ulosonic acid.

Many early molecular events in symbiotic infection have been documented, although factors enabling Rhizobium to progress within the plant-derived infection thread and ultimately survive within the intracellular symbiosome compartment as mature nitrogen-fixing bacteroids are poorly understood. Rhizobial surface polysaccharides (SPS), including the capsular polysaccharides (K-antigens), exist in close proximity to plant-derived membranes throughout the infection process. SPSs are essential for bacterial survival, adaptation, and as potential determinants of nodulation and/or host specificity. Relatively few studies have examined the role of K-antigens in these events. However, we constructed a mutant that lacks genes essential for the production of the K-antigen strain-specific sugar precursor, pseudaminic acid, in the broad host range Rhizobium sp. NGR234. The complete structure of the K-antigen of strain NGR234 was established, and it consists of disaccharide repeating units of glucuronic and pseudaminic acid having the structure -->4)-beta-d-glucuronic acid-(1-->4)-beta-5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid-(2-->. Deletion of three genes located in the rkp-3 gene cluster, rkpM, rkpN, and part of rkpO, abolished pseudaminic acid synthesis, yielding a mutant in which the strain-specific K-antigen was totally absent: other surface glycoconjugates, including the lipopolysaccharides, exopolysaccharides, and flagellin glycoprotein appeared unaffected. The NGRDeltarkpMNO mutant was symbiotically defective, showing reduced nodulation efficiency on several legumes. K-antigen production was found to decline after rhizobia were exposed to plant flavonoids, and the decrease coincided with induction of a symbiotically active (bacteroid-specific) rhamnan-LPS, suggesting an exchange of SPS occurs during bacterial differentiation in the developing nodule.

A novel transposon for functional expression of DNA libraries.

Environmental DNA libraries are important sources for novel biocatalyst genes but activity screening for relevant enzymes is often inefficient. Therefore, we have constructed the transposon MuExpress which randomly integrates in vitro into existing bacterial artificial chromosome (BAC) or cosmid libraries and permits the inducible expression of its flanking regions in both directions. Furthermore, this transposon allows the bidirectional sequencing of the respective clones starting from unique primer binding sites.