INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The structure of microbial communities from many environmental samples is highly complex and diverse. A recent study has estimated that 1 g of soil may contain up to 4,000 different species (33). The complexity of these communities not only is intriguing but also presents a challenge to biotechnology. Current estimates indicate that less than 1% of the microorganisms present in many environments are readily culturable (1). In fact, most of the species in many environments have never been described, and this will not be possible until new culture technologies are developed. Many approaches currently used to explore the diversity and the potential of microbial communities are biased because of the limitations of cultivation methods (3). The classical cultivation techniques require that the different microorganisms derived from an environmental sample be cultured on an appropriate growth medium and then separated until individual clones are isolated. Separation of bacterial communities and growing them on different media, however, results in the loss of major portions of the microbial community, because of the different growth requirements of the many different microbes (3, 23). In addition, this approach is time-consuming and labor-intensive. To overcome difficulties and limitations associated with cultivation techniques, several methods that are DNA based and that bypass cultivation techniques have been developed (15, 25, 31-36). One technique has been very successfully used to construct different environmental DNA libraries and screen for enzymes which can be used in biotechnological processes (15). However, one of the drawbacks of this method is that only small DNA molecules can be cloned from environments. Thus, this technique is limited to the analysis of single genes and consequently precludes screening for metabolic activities encoded in operons or gene clusters. Also, while this paper was under review, Rondon et al. reported the cloning of large DNA fragments directly isolated from one soil type using bacterial artificial chromosomes (25). Although only DNA from one specific soil was isolated and cloned, the method might still cope with the difficulties associated with isolation of DNA from soil when different soils are tested.

Because of the known difficulties associated with the construction of DNA libraries with DNA directly derived from environmental samples, we have chosen to isolate DNA for the construction of cosmid libraries from enrichment cultures. As an example, we used enrichment cultures to select for microorganisms producing high amounts of biotin. DNA was isolated from the enrichment cultures and used to construct cosmid libraries with inserts of >30 kb. The genetic diversity and potential of the different libraries were assessed by screening for biotin biosynthesis operons. This screening was possible because in Escherichia coli and many other gram-negative bacteria, the genes associated with biotin biosynthesis are located in two linked but divergently transcribed operons, which are controlled by a single operator (8). In E. coli, biotin is biosynthesized through the following intermediates: pimeloyl coenzyme A (pimeloyl-CoA)  7-keto-8-aminopelargonic acid (KAPA)  7, 8-diaminopelargonic acid (DAPA)  dethiobiotin  biotin (Fig. 1). Six structural genes and one regulatory element are involved in this biosynthetic pathway; five of the genes (bioA, bioB, bioC, and bio D) are located in a cluster at 17.5 min on the genetic map, forming a biotin operon (2, 8). The bioF, bioA, and bioD genes encode KAPA synthetase, DAPA aminotransferase, and dethiobiotin synthetase, respectively. An enzyme encoded by the bioB gene is involved in the conversion of dethiobiotin to biotin, and the bioC gene is thought to be involved in pimeloyl-CoA synthesis (5, 8, 9, 18, 22, 24).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   (A) E. coli biotin biosynthesis pathway; (B) genetic organization of the E. coli biotin biosynthesis operon.

In this work we have isolated seven cosmid clones, each carrying a different biotin biosynthesis operon derived from environmental consortia. The cosmid clones were characterized on a molecular and physiological level. Although classical enrichment cultures have been widely used for the isolation of microbes with desirable traits, this is the first study to use this approach on a molecular basis, allowing the direct isolation, cloning, and comparative analysis of loci organized in operons or clusters, from diverse microbial niches.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains, plasmids, growth conditions, and environmental samples used in this study. Microbiological materials used in this work are listed in Table 1. E. coli was grown at 37°C on Luria-Bertani medium (28) supplemented with appropriate antibiotics. Enrichment cultures were inoculated with 2.0 g of the different environmental samples (Table 2) into 1-liter flasks containing 100 ml of M9 medium (28) supplemented with 6.5 U of avidin and glucose (10 mM) as the sole carbon source. Except for the enrichment culture derived from horse excrement (Table 2), all cultures were grown at 30°C. Enrichment cultures were incubated on a shaker (150 rpm) for 2 days, pelleted, and used for construction of the DNA libraries. Growth was monitored as optical density at 600 nm (OD 600). The enrichment culture inoculated with horse excrement was grown at 37°C for 24 h.

Molecular techniques. DNA isolation from the enrichment cultures was done as described previously (32), with minor modifications. After pelleting, bacteria were resuspended in Tris-EDTA (TE)-sucrose (20% [wt/vol]) buffer and lysed in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 100 mM Na₂HPO₄ and 1.5 M NaCl, 1% [wt/vol] sodium dodecyl sulfate) for several hours. RNA was degraded with RNase A (10 mg/ml). The resulting DNA extracts were incubated with protease and sarcosyl (5%, [wt/vol]) in TE buffer overnight. Total genomic DNA was then purified repeatedly with chloroform-phenol (1:1, [vol/vol]) and then once with chloroform. DNA was then dialyzed against 2 liters of TE buffer at 4°C overnight. Finally, an aliquot of the DNA was analyzed on a 0.8% agarose gel to ensure that the DNA was not degraded.

Construction of cosmid libraries from the enrichment cultures. Cosmid libraries containing DNA from the various cosmid libraries were prepared in pWE15 (Stratagene, La Jolla, Calif.) using standard protocols (28). DNA fragments (30 to 40 kb) obtained after partial Sau3A digestion were ligated into BamHI restriction sites of the cosmid vector. Phage packaging mixes were obtained from Stratagene, and infection of E. coli VCS257 was performed according to the manufacturer's protocol. Vectors used for subcloning are listed in Table 1; when required, gaps in the DNA sequences were filled by PCR. Automated DNA sequencing was performed using an ABI377 DNA sequencer and dye terminator chemistry following the manufacturer's instructions.

Biotin measurements in culture supernatants. For biotin measurements, use either the Lactobacillus plantarum growth assay (7) or a competitive assay enzyme-linked immunosorbent (ELISA) (4), with some modifications. For this purpose, microtiter plates were coated with anti-rabbit immunoglobulin G at a dilution of 1:5,000 in phosphate-buffered saline (PBS) for 2 hours; washed and treated with blocking solution (vitamin free casein) for 1 h, and then washed with PBS. Extravidin-alkaline phosphatase conjugate (Sigma, Heidelberg, Germany) was added to the plates in a 1:20,000 dilution in PBS-Tween (0.025% [vol/vol]); 100-µl aliquots of the samples were added to the wells, and the microtiter plates were incubated at 37°C for 30 min. Serial dilutions of the various samples were performed prior to the tests, and a standard with known biotin concentrations was included in each microtiter plate. After repeated washing of the microtiter plates with PBS, substrate buffer (Tris-HCl, 100 mM; NaCl, 100 mM; MgCl₂, 50 mM; p-nitrophenylphosphate, 2 mg/ml [pH 9.5]) was added, and color development was recorded at 405 nm in a microplate reader.

Nucleotide sequence accession numbers. The nucleotide sequences obtained have been deposited at GenBank; accession numbers are listed in Table 4.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Enrichment cultures. The enrichment cultures were inoculated with samples derived from five microbial environments (Table 2). The first sample was from a forest soil with an extremely high humic acid content, collected near Goettingen, Germany; the second sample was soil collected from an agricultural site near Goettingen. The third sample consisted of horse excrement collected from a meadow near Goettingen; this sample was likely already enriched with a dominant population of enteric bacteria. The fourth sample was collected from a beach in the north of Greece, near Kavalla, and the fifth sample was collected on Mount Hood, in Oregon (Table 2).

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Scheme of the enrichment and cloning strategy used in this study. The samples were taken from five different microbial environments (an agriculture soil, a forest soil, horse excrement, volcanic soil, and sandy soil).

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Dot blot hybridization of DNA extracted from environmental samples and enrichment cultures. (A) Nylon membrane with DNA directly isolated from the HE sample and DNA from a corresponding HE enrichment culture, which was grown in the presence of avidin; 780 ng of DNA was spotted per dot, and hybridizations were done with the E. coli biotin biosynthesis genes as a DNA probe. (B) Nylon membrane loaded with DNA which was isolated from two different enrichment cultures inoculated with soil from an agricultural site (AS); one of the cultures was grown in the presence of added avidin, while no avidin was added to the other culture. DNA was extracted after 48 h of growth, and 930 ng of DNA was spotted per dot onto the nylon membranes. Hybridizations were performed overnight under high-stringency conditions at 68°C employing the bio genes on pCosAS1 as a DNA probe.

Construction of cosmid DNA libraries from enrichment cultures. To identify biotin biosynthesis genes associated with the three enrichment cultures, DNA was isolated from bacterial pellets by direct lysis. The DNA was size fractionated to fragments ranging in size from 30 to 40 kb and ligated into the cosmid vector pWE15 (see Materials and Methods). After packaging of the DNA and transfection into the E. coli host strain, the three libraries (HE, AS, and FS) ranged in size from 6,000 to 35,000 clones. The quality of the libraries was ensured by estimating the insert sizes in 70 randomly picked clones; the average insert size was approximately 30 to 40 kb, and 98% of the clones analyzed contained inserts. In addition, restriction analysis of randomly chosen cosmid clones revealed high diversity of the DNA fragments cloned into pWE15.

Screening for DNA fragments containing bio operons. To assess the biotin biosynthesis capabilities of the three environmentally derived cosmid libraries, cosmid DNA from the recombinant E. coli clones was extracted and used to transduce the E. coli biotin-auxotrophic strain ATCC 33767 (Fig. 2). This strain carries a deletion in the region of the bio operon, ranging from the lambda attachment site to the uvrB gene, and consequently is not able to synthesize biotin unless complemented by a complete biotin biosynthesis operon. After transduction, cells were transferred to M9 medium in the absence of biotin, allowing only growth of cells containing transduced DNA complementary to the host E. coli auxotrophic strain. Bacteria growing in liquid medium were spread onto agar plates and incubated overnight. From these possible bio⁺ clones, DNA was extracted and used to retransduce E. coli strain ATCC 33767. This procedure was repeated twice to avoid the isolation of false-positive clones. Only bio⁺ clones growing after the second transduction were subject to subcloning and further molecular analysis (Fig. 2). Employing this strategy, we isolated seven different cosmid clones which appeared to contain complete biotin synthesis operons and complemented the E. coli auxotrophic strain when grown on plates and in liquid medium. A DNA restriction analysis ensured that each clone was unique. Four of these seven cosmid clones carrying the different bio operons were subject to a detailed molecular and physiological analysis. The clones isolated, named pCosHE1 and pCosHE2, originated from the HE library. The cosmid clones pCosAS1 and pCosFS1 were isolated from the AS and FS libraries, respectively.

Biotin biosynthesis capabilities and growth characteristics of the bio cosmids. To examine the biotin biosynthesis capabilities of the isolated cosmid clones, we measured growth and biotin synthesis capabilities of cosmid clones pCosHE1, pCosHE2, pCosAS1, and pCosFS1 under defined conditions. Because succinate and glucose as sole carbon sources are known to affect biotin synthesis, we chose to add these carbon sources to the defined medium. Data were compared to growth and biotin synthesis capabilities of the E. coli K-12 wild-type isolate. For the E. coli control strain, growth on succinate resulted in a 5.8-fold-higher biotin production than growth on glucose (Table 3). Growth on succinate also stimulated biotin biosynthesis for two of the clones, pCosHE1 and pCosHE2; the observed increase in biotin production was highest for pCosHE2 (90-fold), followed by pCosHE1 (4.1-fold). For pCosAS1 and pCosFS1, however, biotin production was highest after growth on glucose.

Molecular characterization of the different cosmids conferring biotin biosynthesis capability. To further characterize the four bio clones, DNA was isolated, restricted with different enzymes, and subject to partial sequencing. Restriction analysis and shotgun sequencing confirmed that the insert sizes in pWE15 were approximately 30 to 40 kb and allowed a detailed mapping of the cosmids (Fig. 4). A partial sequencing of DNA derived from the cosmids in combination with DNA hybridizations identified and located four different biotin biosynthesis operons on the different cosmids. Figure 4 and Table 4 summarize the identified genes and similarities observed after comparison of the deduced amino acid sequences obtained with those available in the National Centre for Biotechnology Information databases. The organization of the bio genes identified on the four cosmids appeared to be the same as four for most gram-negative bacteria (e.g., E. coli). This finding is based on DNA sequencing and DNA hybridization studies. The deduced amino acid sequences of the bio genes identified by sequencing on pCosHE1 revealed highest similarities with the corresponding proteins in Erwinia herbicola. Identities observed for bioA and bioB were 84 and 85%, respectively. Downstream of the bio operon, the moaA-BCDE operon was identified. Comparison of the deduced amino acid sequences showed an overall 80% identity with MoaA-BCDE in E. coli. In addition, several other genes pCosHE1. (uvrB, elsB, elsC and hutH) were identified on. The deduced amino acid sequence of hutH was 81% identical to the deduced amino acid sequence of the Pseudomonas putida HutH. Also, we observed on this cosmid genes coding for proteins involved in DNA transfer and pilus assembly, as well two different antibiotic resistance markers (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Restriction maps of the central parts of the different biotin cosmids isolated in this work; arrows indicate locations and directions of transcription of the identified ORFs on the different cosmids. (A) pCosHE1 restriction map of the bio cosmid derived from horse excrement showing highest homologies to genes of E. herbicola; (B) pCosHE2 bio cosmid isolated from an enrichment culture derived from the same source as in panel A but highly similar to E. coli; (C) pCosAS1 restriction map of a bio cosmid isolated from an agricultural soil; (D) pCosFS1 restriction map of a bio cosmid isolated from a forest soil enrichment culture. The bio genes identified in panels C and D show highest similarities to E. herbicola and S. marcescens, respectively. Observed similarities for selected ORFS are listed in Table 4, together with the assigned GenBank accession numbers. For genes of pCosHE1 and of pCosFS1, only partial sequences were obtained during the shotgun sequencing approach. DNA restriction enzymes: N, NcoI, B, BamHI; E, EcoRI; Sm, SmaI; P, PstI; K, KpnI; H, HindIII; Sc, SacI.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this work we have developed an approach utilizing enrichment technology, which allows the isolation and comparative analysis of gene clusters or operons from diverse microbial consortia, as opposed to previous studies with the principal goal to obtain variants of one specific gene of interest. As a first step, our technique relies on the preparation of an enrichment. The technique of enrichment culture offers a powerful tool, with an intriguing potential to select for microbes with traits useful for biotechnological applications. In general, enrichment cultures have been successfully used to screen for single microbes or consortia with diverse catabolic capabilities. Specifically, enrichment cultures have been employed to select for microbes capable of degrading toluene (17) or phthalate (20), utilizing methane (6), or dechlorinating and degrading diverse aromatic compounds (10, 19, 26). The technique has also been successfully applied to enrich for microbial consortia or individual strains with enhanced capabilities for cellulose degradation (27) or scyllo-inosamine degradation (11). Nevertheless, while it is well known that enrichment cultures result in the loss of major portions of the microbial populations associated with any given environment (3), it is still possible for the enriched cultures to contain a highly diverse population of organisms (11, 19).

A major reason for the isolation of DNA from enrichment cultures arose from the difficulties associated with the isolation of high-molecular-mass DNA from microbial habitats. Although during the last decade different approaches for the isolation and purification of bacterial DNA from a variety of environments have been reported, the vast majority of the techniques described are not suitable for the efficient construction of libraries with large inserts. Most of the available techniques can be grouped in two major classes: first, the isolation of DNA after direct lysis of the microorganisms in the presence of the organic soil matrix; and second, the isolation of the DNA after separation of the bacteria from the soil matrix (16). However, one difficulty with both of these methods is that the purified DNA is often contaminated with polyphenolic compounds, which are copurified with the DNA. The contaminating compounds are difficult to remove, and it is well known that the polyphenols interfere with enzymatic modifications of the isolated DNA (16). As a result, the construction of environmentally derived DNA libraries with large inserts is almost impossible due to the poor quality of the isolated DNA. In two recently published model studies, the isolated DNA has been used for the construction of DNA libraries leading to the isolation of genes with desirable industrial traits (15, 25). Our attempts to construct DNA libraries with large inserts following the protocol published by Henne et al. (15), i.e., via extraction of the DNA directly from the different environmental samples failed. Therefore, we have developed a strategy that allows us to isolate highly pure DNA from microbial consortia after enrichment culture and that would allow construction of DNA libraries with insert sizes larger than 30 kb (Fig. 2). The construction of libraries with sufficiently large inserts was essential to allow screening for the presence of complete bacterial operons encompassing 5 to 6 kb in size.

In this work, we inoculated five enrichment cultures with environmental samples, four from soil and one from horse excrement (Fig. 2; Table 2). Because of the high selection pressure caused by the presence of the biotin-complexing agent avidin in the medium, bacteria able to synthesize high amounts of biotin were primarily enriched. Not much is known about the biotin biosynthesis rates of bacteria in a natural environment, and it can only be assumed that most microorganisms in microbial habitats are prototrophs. Only data on actual rates of biotin production of wild-type isolates in pure cultures are available. In our studies E. coli K-12 produced approximately 8 pg of biotin/ml in 24 h on a defined medium (Table 3); a Klebsiella pneumoniae isolate produced 350 pg/ml. Two typical soil bacteria, Rhizobium meliloti 1021 and Ralstonia eutropha H16, however, produced in the same time under the same conditions less than 10 pg of biotin/ml, which was below the detection limit.

DNA extracted from the enrichment cultures was sufficiently pure to allow the construction of cosmid libraries harboring large (>30-kb) inserts. Although the applied strategy likely resulted in the loss of major portions of the populations associated with each of the microbial communities (Table 2), we were able to use this method to isolate seven different cosmids complementing an E. coli bio mutant. Although we have not characterized all of the bio cosmids in detail on a molecular level, we can assume that each of the seven isolated cosmid clones carries a complete biotin biosynthesis operon; this is primarily because the E. coli auxotrophic mutant used can grow only if a complete bio operon is present. Four of the seven isolated cosmids conferring the ability to synthesize D-biotin were subject to a detailed study. Amounts of biotin produced were measured, and the results demonstrated that each of these bio clones produced significant amounts of biotin, indicating the successful expression of the identified operons.

DNA sequencing revealed that one of the isolated biotin biosynthesis operons was highly similar to the known E. coli bio operon (Table 4). The other identified biotin synthesis genes showed highest similarities to corresponding genes from E. herbicola and S. marcescens. Both microorganisms are closely related and belong to the family Enterobacteriaceae. Although the observed similarities were surprisingly high for several of the identified genes, we have no evidence indicating from which species the bio cosmids were derived. The isolated biotin synthesis operons appear to have the same bidirectional organization as the biotin biosynthesis operons of E. coli and other closely related organisms (Fig. 4) (2, 8, 12, 14). This suggests the possibility that the conserved organization of these genes is important for the synthesis of biotin or for its regulation. However, general rules for the organization of bio genes cannot be made. Finally, it must be kept in mind that our screen approach based on the complementation of an E. coli biotin auxotrophic mutant lacking the entire bio gene cluster allowed the isolation only of biotin biosynthesis operons whose promoters could be recognized by the host strain. This explains why the total number of bio⁺ clones isolated form an average of 6,000 to 30,000 screened clones is relatively low. Thus, we can only speculate that the use of a different screening system (e.g., a biotin-auxotrophic Bacillus strain) would have resulted in the isolation of bio genes closely related to gram-positive bacteria. Also the use of a mobilizable expression vector might have increased the number of resulting bio clones.

Although the isolated biotin biosynthesis operons were similar in structure, major differences were observed within the flanking regions (Fig. 4; Table 4). While in E. coli the galactose operon is upstream of the bio operon (2), we found the histidine utilization operon (hut) upstream of bioA in two of the characterized operons (Fig. 4). The presence of the hut operon upstream of the biotin biosynthesis genes has been described for Salmonella enterica serovar Typhimurium and K. aerogenes (12). However, none of the isolated bio cosmids shows a sufficient similarity to the bio genes isolated from S. enttrica serovar Typhimurium and K. aerogenes to warrant the claim that it was isolated from one of those organisms. In addition to the hut operon, we have identified the moaABCDE genes in pCosHE1, coding for genes involved in the synthesis of molybdopterin cofactors. Those genes are commonly found downstream of bio operons in gram-negative bacteria. The moa genes and the hut genes are two more examples of genes that are organized in a gene cluster and which we could isolate as a whole with the combined cloning and enrichment technique used in this work. Consequently, this technique will be a valuable tool for the isolation of operons or conserved gene clusters from environmentally derived microbial consortia.