The use of a PCR cloning and screening strategy to identify lambda clones containing the hupB gene of Anabaena sp. strain PCC7120

ELSEVIER

Journal of Microbiological

Methods

27 (1996)

175-182

Journal ofMicrobiological Methods

The use of a PCR cloning and screening strategy to identify lambda clones containing the hupB gene of Anabaena sp. strain PCC7120’ Jyothirmai

Gubili”‘b, Dulal Borthakur”‘”

“Department of Plant Molecular Physiology, University of Hawaii, Honolulu, HI 96822, USA bDepartment of Microbiology, University of Hawaii, Honolulu, HI 96822, USA Accepted 26 August 1996

Abstract Two lambda clones containing the hupB gene of Anabaena sp. strain PCC7120 were isolated from a genomic DNA library using a cloning and screening strategy which involved the polymerase chain reaction (PCR). Two highly conserved stretches of amino acid5 were identified within the HupB sequences of various organisms and two degenerate PCR primers were synthesized based on these sequences. Upon amplification of the Anabaena genomic DNA using these primers, a 27%bp DNA fragmenlt was obtained. By sequencing this PCR fragment it was established that this was a part of the Anabaena hz@? gene. Based on this sequence, two high-stringency PCR primers were synthesized that were used to isolate two lambda clones containing the hupB gene by PCR-screening of an Anabaena genomic library. By using a modified screening procedure the two lambda clones were directly isolated from those plates which appeared positive in PCR mass-screening and in which the plaques were cross-contaminated during the mass screening. Keywords:

PCR cloning; hupB gene; Anabaena;

Hydrogenase

1. Introduction Anabaena sp. strain PCC7120 is a filamentous photosynthetic cyanobacterium capable of aerobic nitrogen fixation. In the absence of combined nitrogen, such as ammonia, Anabuenu filaments develop certain specialized cells for nitrogen fixation, called heterocysts, at regular intervals of lo-12 vegetative cells [l]. During nitrogen fixation in the heterocysts, hydrogen is produced as an obligate by-product

*Corresponding author, Tel: +l 808 9566600, fax: +I 808 9563542; e-mail: dulal@)hawaii.edu ‘Journal Series no. 420’7 of the Hawaii Institute of Tropical Agriculture and Human Resources.

0167-7012/96/$15.00 Cclpyright PII SO167-7012(96)00947-5

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which is recycled by a membrane-bound uptake hydrogenase. The electrons so produced flow through a respiratory chain and can be coupled to ATP synthesis [2]. The overall effect of uptake hydrogenase is to increase the metabolic efficiency of nitrogen fixation. The uptake hydrogenase has been characterized in several nitrogen-fixing bacteria such as Brudyrhizobium juponicum, Rhizobium leguminosurum, Azotobacter chroococcum, Azotobucter vinelundii and Rhodobucter cupsulutus [reviewed in [3-611. These enzymes are membranebound, nickel-containing proteins with two polypeptide subunits of approximately 35 and 65 kDa. Besides these two subunits there are other accessory enzymes ‘which are involved in the processing and

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maturation of the hydrogenase enzymes. The HupB is one such enzyme which is involved in nickel incorporation into the hydrogenase involving GTP hydrolysis [7]. Hydrogenase enzymes have also been characterized in non-nitrogen-fixing organisms, such as Escherichia coli and Desulfovibrio spp. [3,4]. The function of hydrogenase in these organisms is to catalyze hydrogen oxidation coupled to energy conserving reduction of electron acceptors like fumarate, nitrate, sulfate and CO, [6]. Hydrogenase can also directly reduce reductant pools such as ferredoxin, pyridine nucleotides and flavins that normally participate in biosynthetic reactions. Alternatively, reduction of these electron carriers may require a reverse electron flow through a membrane-bound electron transfer chain. Electrons from H2 can replenish the electron transport chain resulting in ATP synthesis. The transfer of a pair of electrons from H, to an electropositive acceptor such as OZ releases sufficient energy to support the synthesis of several molecules of ATP. The genes involved in hydrogen metabolism (hyp and hup) have been cloned and sequenced in a number of organisms [5]. Twenty genes have been identified in the Rhodobacter capsulatus hup gene cluster [8]. The two genes encoding the small and the large subunits of the Anabaena uptake hydrogenase have been cloned and sequenced recently [9]. Our earlier attempts to clone the hup genes from Anabaena using Bradyrhizobium hup genes as probes were unsuccessful, presumably because hup genes of this organism do not share high DNA sequence homology with other bacteria. However, we hypothesized that there may be small stretches of conserved sequences within the hup genes of Anabaena where the homology with other bacteria may be sufficiently high, which should allow us to isolate these genes by screening a genomic library. We used a PCR cloning and screening strategy based on conserved stretches of DNA sequences to isolate the hupB gene of Anabaena sp. strain PCC7120. Two highly conserved stretches of amino acids were identified within the amino acid sequences of various organisms upon sequence comparison and two degenerate PCR primers were synthesized based on these sequences. Upon amplification of the Anabaena genomic DNA using these primers, we obtained a 278-bp product as expected. By cloning

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and sequencing the PCR fragment, it was established that this fragment is a part of the Anabaena hupB gene. Based on this sequence we made two highstringency PCR primers which were used to isolate clones containing hup genes by PCR-screening of an Anabaena genomic library. The isolation of these clones involved two steps: (i) identification of a plate containing 100 plaques by PCR screening of the lambda lysates from 100 plates, and (ii) using the identified plate for PCR screening of individual plaques to isolate the desirable clones. We show here that enough phages were left in the individual plaques after the first elution of the lysate to allow us to identify the positive clones in spite of some cross-contamination of the plaques in these plates during the previous mass-elution of phages.

2. Materials

and methods

2.1. PCR conditions The PCR reactions were done using the Gene Amp PCR system 2400 (Perkin Elmer). Standard PCR reactions were done in a final volume of 50 ~1, which included 100 ng of sample DNA, 200 PM of each of dATP, dCTP, dGTP and dTTP, 0.2 PM of each specific primer, 2.5 mM MgCl, and 1.5 U of 7’aq polymerase (Promega, Madison, WI). Thermal cycling was done as follows for 30 cycles: denaturation for 1 min at 94°C primer annealing at 58°C and extension at 72°C for 3 min. For screening the lambda library, a 2-,~~1 aliquot from the lambda mixture eluted in phage buffer was used as the template and the PCR conditions were as follows. The template was denatured at 94°C for 15 min followed by heating at 80°C for 45 min. Reactions were then done under the same conditions as mentioned above after adding all the reagents. PCR products were electrophoresed on 1.5% agarose gels and visualized by staining with ethidium bromide. 2.2. Construction

of two degenerate

PCR primers

The hupB genes of A. chroococcum [lo] and the hypB genes of E. cob [ll], R. leguminosarum [12], B. japonicum [13], R. capsulatus [8] and A. vinelandii [ 141 have homology at the deduced amino

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acid level. By aligning the amino acid sequences of these genes, four highly conserved regions were identified between amino acid numbers 128 and 221 as shown in Fig. 1. Two stretches of ten amino acids from regions 1 and 4 were selected and converted into DNA sequences using the most probable codon usage for Anabaev~a (Fig. 1). The sequence of twenty nine nucleotides of the forward strand from region 1 was selected for primer 1 and another sequence of 29 nucleotides from the reverse strand corresponding to r’egion 4 was selected for the reverse primer. These two primers had similar GC content and melting temperatures and were not homologous to each other. Primers were synthesized in the Biotechnology and Molecular Biology Instrumentation Facilities, University of Hawaii, Honolulu. 2.3. DNA preparation

and Southern

hybridization

Growing of Anabaena cultures, genomic DNA isolation and Southern hybridizations were done as previously described [15] except that DNA probes were labeled by random priming using the dioxigenin labeling and detection kit from Boehringer Mannheim, Indianapolis, USA. 2.4. Construction

qf the lambda library

A genomic library of Anabaena sp. strain PCC7 120 was constructed by inserting lo- 16 kb partially digested SauIIIA fragments into the BamHI

srmer

<

rrw____u_(.Lw3

Borraird

I

oIx”T1m.mxFrm=mcA==m1 Reverse

Fig. I. Construction of two degenerate PCR primers for amplification of a section of the Anabaenn hupB gene. The deduced HupB amino acid sequence of Azotobacter chroococcum (AC), and HypB sequences of E. coli (EC:), A. vinefandii (Av) and R. capsularus (Rc) were aligned and four highly conserved regions were identified between amino acid 128 and 221 corresponding to A. chroococcum sequences. The forward and the reverse degenerate primers were synthesized using the most probable codon preferences for Anabaena.

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site of the lambda Gem1 1 vector (Promega, Madison, WI). The library of approximately 15 000 clones was amplified in E. coli strain LE392.

2.5. Plating and screening

of the lambda library

The primary screening of the lambda library was done by some modification of the method described by Amaravadi and King [16]. The library was plated in ten 85mm plates each containing approximately 3000 plaques. The phages were soaked with 2.5 ml of phage buffer (50 mM Tris-HCl. pH 7.5, 100 mM NaCl and 8 mM MgSO,) by incubating for 2 h at 37°C. One milliliter of phage buffer containing the phages was collected from each plate and centrifuged in a microfuge to remove the debris and stored at 4°C. A 2-~1 aliquot from each phage stock was used for PCR reactions. One positive aliquot was selected and further plated in ten plates to obtain approximately 500 plaques per plate. These plates were screened as above and a positive aliquot was selected. This aliquot was plated in 100 plates each containing approximately 100 plaques. The phages were eluted with phage buffer as described above and transferred separately to 100 1.5-ml tubes. Ten microliter aliquots from these 100 samples were combined in groups of five in 20 tubes, mixed and screened by PCR. A positive mixture was selected and the five original tubes, from which this mixture was made, were individually screened by PCR. In this way a positive sample was identified which originated from a plate containing 95 plaques. The 95 plaques from the positive plate were individually picked using Pasteur pipettes and transferred into 95 test tubes containing 100 ~1 phage buffer. Note that the phages from this plate were previously eluted using 2.5 ml phage buffer and therefore the individual plaques are expected to be cross-contaminated to some degree during the mass elution. Five microliter aliquots from these 95 plaque samples were combined into groups of five in 19 tubes and screened by PCR. The sample that appeared positive with a strong band was identified and its five component plaques were further screened to identify a positive single plaque sample. Several dilutions ,of this sample were plated to obtain isolated individual plaques. Ten plaques from one of these

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plates were individually to identify the positive 2.6. Sequence

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picked and screened by PCR clone.

comparison

DNA and deduced amino acid sequences were compared with the data base using the Experimental BLAST Network Service [17].

3. Results 3.1. Cloning and sequencing of a 278-bp hupB fragment of Anabaena generated by PCR using two degenerate primers A 278-bp PCR fragment was generated by using the two degenerate primers and Anabaena DNA as the template (Fig. 2). The same results were obtained when a MgCI, concentration of 1.5, 2.5 or 3.5 mM was used in the PCR reaction. These primers generated a faint band with E. coli genomic DNA but not with Spirulina platensis strain pacifica DNA as the template (data not shown).

DM

ABC

278

bp

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This fragment was cloned into the pGEM-T vector and sequenced. The nucleotide sequence of this fragment excluding the two 29-bp regions covered by the primers on both sides is shown in Fig. 3A. The DNA sequence showed 54% homology with and Bradyrhizobium japonicum Rhizobium leguminosarum hypB sequences. When this sequence was translated into amino acid sequences, the deduced amino acid sequences showed 54-62% identity with different HypB sequences (Fig. 3B). High homologies of 78 and 89% were observed in the sequences corresponding to conserved regions 2 and 3, respectively, as shown in Fig. 1. 3.2. Construction of two high stringency PCR primers specific for the hupB fragment of Anabaena The two degenerate primers also amplified a faint PCR fragment with the E. coli genomic DNA as the template. Therefore, it was not possible to use them for PCR screening of lambda clones containing the Anabaena hupB gene. Based on the 220-bp Anabaena hupB sequence, two 23-nucleotide primers were synthesized for high stringency PCR specific for the Anabaena hupB DNA (Fig. 3A). These primers have similar GC content and melting temperature and did not have homology to each other. When they were used against Anabaena genomic DNA in a PCR reaction, a 166-bp fragment was observed, as expected (Fig. 4). This fragment was purified from the gel and sequenced in both direc-

331 bp

Fig. 2. The 278-bp generated by using the two degenerate primers against the Anabaena genomic DNA. The PCR fragment was generated when the concentration of M&l, in the reaction was 1.5 mM (lane A), 2.5 mM (lane B) or 3.5 mM (lane C). A 331-bp fragment was obtained when the plasmid containing the cloned 278-bp PCR fragment was digested with AarII and PsrI (lane D). The pGEM marker is shown in lane M.

Fig. 3. A. DNA sequence of the 278-bp PCR fragment excluding the two degenerate primer regions in both ends. The sequences of the two high-stringency primers are shown within boxes. B. The comparison of the deduced amino acid sequences of the 220-bp Anabaena hupB segment with HypB amino acid sequences from R. leguminosarum (Rl), R. capsulalus (Rc), A. chroococcum (AC), A. Idnelandii (Av), E. coli (EC) and B. japorticum (Bj).

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-166bp

166bp-+

166 bp--+ + 166bp

Fig. 4. PCR screening of ii lambda library of Anabaena genomic DNA using two high-stringency primers. Panel (a): the two high-stringency primers amplified a 166~bp fragment when Anabarna 7120 genomic DNA (lane A) or 2 ~1 of the lysate from the library was used as the template (lane B). They did not generate a fragment with E. coli genomic DNA (lane C), plasmid pBR322 (lane D), or lambda DNA (lane E) as the template. No template DNA was used in the negative control (-). The pGEM marker is shown in lane M. Panel (b): Identification of two positive pools (lanes M and P), each comprising five plates with approximately 100 plaques each. Lysate from a pool of 3000 plaques that was positive in the previous round of screening was used as the positive control. The lysates from individual plates from these positive pools were screened and a positive plate was identified (not shown). Panel (c): Screening of individual plaques isolated from a positive plate in which the plaques were cross-contaminated during PCR mass-screening. A bright band is seen in lane F which indicates a ‘true positive’. Other lanes show a light band due to cross contamination of plaques during mass-screening of the plates in previous step. Panel (d): The contaminal.ed phages from the positive plaque were plated and the isolated plaques were individually screened. 50% of these plaques were positive (lanes A, B, E, H and J).

tions using the same two primers. The sequence showed that this fragment was the inner segment of the previous fragment generated by using the two degenerate primers. When the high-,stringency primers were used against the genomk DNA of E. coli in a PCR reaction, they did not generate a fragment. However, when ti 2 ~1 lysate of the lambda library of the Anabaena genomic DNA was used as the template, it amplified a DNA fragment of expected size, sug-

gesting that the lambda library had clones containing the Anabaena hupB gene (Fig. 4). 3.3. Isolation of two lambda clones containing hupB gene of Anabaena

the

The lambda library of Anabaena genomic DNA was screttned using PCR as described in Section 2. Five plates were identified each containing approximately 3000 plaques that appeared positive. When

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the lysate from one of these plates was used for the secondary screening, one plate containing approximately 500 plaques was identified as a positive. When aliquots of the lysate from this plate were plated in 20 plates each containing approximately 100 plaques, none of these plates was found to be positive. Therefore, we plated 100 plates each containing approximately 100 plaques, among which two plates were found to be positive. The plaques from these two plates were picked individually and finally we identified two lambda clones from two positive plates. These clones were named as AJGI and hJG2. hJG1 and hJG2 could not be distinguished on the basis of restriction pattern with HindIII and HincII (Fig. 5a). There were two Sac1 restriction sites on either side of the BumHI site in lambda GEM1 1. Therefore the insert fragments could be completely cleaved out by this enzyme (data not shown). It was found that both clones contained a 1%kb insert DNA. When the 166-bp PCR fragment generated by the two high stringency primers was used as the probe, it hybridized with a 3.6-kb Hind111 fragment of these clones and a fragment of the same size in the HindIII-digested genomic DNA of Anabaena 7120 (Fig. 5b). The same probe hybridized with a 2.0-kb HincII fragment of these clones. The PCR

Fig. 5. Panel a: Common DNA fragments in AJGl and AJG2. The two lambda clones were cleaved with restriction enzymes and DNA fragments were separated on a 0.8% agarose gel. Panel b: Southern hybridization of AJGl and AJG2 DNA with the 166-bp PCR fragment. The gel in panel ‘a’ was transferred to a nylon membrane and hybridized with dioxigenin-labeled 166-bp PCR fragment.

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fragment hybridized with a 20-kb fragment in the EcoRI-digested Anabaena genomic DNA. Only a part of this 20-kb fragment was cloned in hJG1 and hJG2.

4. Discussion Plaque hybridization is the most widely used method for screening lambda libraries. However, when the frequency of the desirable clone is low, it is often difficult to identify clones by plaque hybridization. Our attempts to isolate a lambda clone containing the Anabaena hup genes by plaque hybridization using non-radioactive labeling method was not successful. When we used the PCR method to detect the presence of the desired clone in the library, we obtained the expected band suggesting that the desired clone was present in the library. Previously, Amaravadi and King [16] used the dilution plating method in combination with PCR to isolate clones from a lambda library. That method had two drawbacks: firstly, it is difficult to identify positive plates containing a few plaques. Secondly, the individual plaques were not screened by picking them directly from the positive plate. The phages collected from the positive plate had to be re-plated to screen individual plaques. The frequency of the desirable plaques might be changed during re-plating of the phages collected from the positive plate. We unsuccessfully screened several hundred individual plaques obtained by plating the lysate of a positive plate containing 100 plaques. Later, we discovered that the desirable clone could be identified from a positive plate without re-plating the phages. Soaking the plates with 2.5 ml of phage buffer for PCR mass-screening removed only a part of the phage particles from the plaques. The individual plaques of such a soaked plate still contained enough phages to allow the detection of the desirable plaque by PCR. Although the cross contamination due to previous soaking and mass-extraction of plaques from the same plate caused some background effects in the PCR reactions, the real positives showed an intense band and were easy to detect. The isolation of the Anabuenu hupB clone became possible by use of this modified method. We used degenerate PCR primers to clone a part

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of the Anabaena hupB gene. However, the same primers could not be used for screening the library, because the degenerate primers produced a PCR fragment of similar size with the E. coli DNA also. The PCR fragment generated from E. coli was of lesser intensity than that from Anabaena and may contain a part of the hypB gene of E. coli. The high stringency inner primers, on the other hand did not generate a PCR fragment in E. coli and were very specific for Anabtrena DNA. Therefore, it was possible to use these inner primers to identify two desirable clones by PCR screening of the library. The PCR screeniqg strategy described here is a combination of several screening procedures that are generally used to fish out specific genes from among families of similar sequences in higher organisms. Identification of clones by the PCR method is also the corner stone of the human genome project, where pools of clones are subjected to PCR assays to determine the clonle’s position in the existing sequence contigs. Our results show that a combination of screening procedures involving PCR can be used to fish out particularly troublesome sequences, even in bacterial systems. The advantage of the PCR screening method is that extraction of DNA is not required from the phages isolated from each plate. Usually 1 ~1 of the lambda library would contain at least a million clones which should be adequate for detecting the desirable sequence by PCR. We found that cleaning the lysate by passing it through 0.45 ,um pore size filter did not enhance the amo/unt of the PCR product. The removal of the bacterial debris by spinning the lysate in a microcentrifuge is enough to get a good PCR product. The second screening of the lambda library resulted in the identification of a positive plate containing 500 plaques However, in the third screening to identify a positive plate containing 100 plaques, we expected that one out of every five plates would be positive. Surprisingly, we found that only one out of fifty plates was positive. Moreover, after identifying one positive plate with 95 plaques, we plated the lysate of this plate to obtain isolated plaques and screened 500 plaques unsuccessfully to obtain a single positive. We do not know the reason for this unexpected result. It is possible that some of the genes in hJG1 and hJG2 have deleterious effects on

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the E. coli cells infected by them and therefore grew slowly or lysed early to produce fewer lambda particles. There are twenty genes in the hup gene clusters of Rhodobacter [8] and Azotobacter [ 101. It is expected that the homologs of these genes are present in Anabnena also. Recently, Carrasco et al. [9] identified the hupL gene of Anabaena while sequencing genes involved in rearrangement during heterocyst differentiation. The hupL DNA sequence has only 54% homology with the B. japonicum or other hupB genes. At DNA sequence level, other Anabaena hup genes may not have high homology with Azotobacter, Rhodobacter or E. coli hup genes sequences. However, our hypothesis at the onset of this project that there may be short stretches of conserved sequences where the Anabaena hupB gene may have homology with hupB gene sequences of other organisms proved to be correct. The cloned PCR fragment showed homology with. the hup genes of other organisms at both DNA and deduced amino acid sequence levels.

Acknowledgments This work was supported by a grant (DE-FG06 94AL85804) to the Hawaii Natural Energy Institute from the US Department of Energy via the National Renewable Energy Laboratory. We would like to thank Dr. Oskar Zaborsky for useful discussions and Dr. Leslie R. Berger for reviewing the manuscript.

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[6] Voordouw, G. (1992) Evolution of hydrogenase genes. Adv. Inorg. Chem. 38, 397-422. [7] Maier, T., Jacobi, A.. Sauter, M. and A. Bock (1993) The product of h?pvpBgene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotidebinding protein. J. Bacterial. 175, 630-635. [8] Colbeau. A., Richaud. P., Toussaint, B., Caballero, F.J., Elster, C., Delphin, C.. Smith, R.L., Chabert, J. and Vignais, P.M. (1993) Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants. Mol. Microbial. 8, 15-29. [9] Carrasco, C.D.. Buettner, J.A. and Golden, J.W. (1995) Programmed DNA rearrangement of a cyanobacterial hupL gene in heterocysts. Proc. Natl. Acad. Sci. USA 92, 791795. [lo] Tibelius, K.H., Du, L., Tito, D. and Stejskal, F. (1993) The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hupA, hupB, hupY and hupC. Gene 127, 53-61. [ll] Lutz, S., Jacobi, A., Sch1ensog.V.. Bdhm, R., Sawers, G. and A. Bock, A. (1991) Molecular characterization of an operon (hyp) necessary for the activity of the three hydrogenase isoenzymes in Escherichia coli. Mol. Microbial. 5, 123-135.

[ 121 Rey, L., Murillo, J., Hemando, Y., Hidalgo, E., Cabrera, E., Imperial, J. and Ruiz-Argiieso, T. (1993) Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium leguminosarum biovar viciae. Mol. Microbial. 8. 471-481. [ 131 Fu, C., Olson, J.W. and Maier, R.J. ( 1995) HypB protein of Bradyrhizobium japonicum is a metal-binding GTPase capable of binding 18 divalent nickel ions per dimer. Proc. Natl. Acad. Sci. USA 92, 2333-2337. [l4] Chen, J.C. and Mortenson, L.E. (1992) Identification of six open reading frames from a region of the Azotobacter vinelandii genome likely involved in dihydrogen metabolism. B&him. Biophys. Acta 1131, 199-202. [15] Borthakur. D. and Haselkom, R. (1989) Tn5 mutagenesis of Anabaena sp. strain PCC7120: isolation of a new mutant unable to grow without combined nitrogen. J. Bacterial. IO, 5759-5761. [ 161 Amaravadi, L. and King, M.W. (1994) A rapid and efficient non-radioactive method for screening recombinant DNA libraries. BioTechniques 16, 98-103. [17] Altschul, S.F., Gish. W., Miller, W.. Myers, W.E. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403-410.