Use of glnQ as a Counterselectable Marker for Creation of Allelic Exchange Mutations in Group B Streptococci

Efficient allelic exchange mutagenesis in group B streptococci(GBS) has been hampered by the lack of a counterselectable markersystem. Growth inhibition of GBS by the glutamine analog gamma-glutamylhydrazide requires glnQ. We have used this phenomenon to createa counterselectable marker system for efficient selection ofallelic exchange mutants in GBS.

INTRODUCTION

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Introduction

Group B streptococci (GBS) are the leading cause of meningitisand sepsis in newborns in the United States and Western Europe(1). Recent advances have allowed for genetic manipulation ofGBS, including the use of temperature-sensitive (TS) vectorsfor creation of allelic exchange mutations (17). However, creationof allelic exchange mutations in GBS is a laborious processthat sometimes requires replica plating several thousand individualcolonies (H. H. Yim, unpublished data). Creation of such mutationshas been hampered by the lack of a counterselectable markersystem for use with GBS.

We have recently discovered that wild-type GBS do not grow inthe presence of the glutamine analog gamma-glutamyl hydrazide(GGH) and that this inhibition requires the glutamine transportgene glnQ (14). This finding led us to hypothesize that glnQ,when expressed on a plasmid in the background of a parent strainwith a deletion in glnQ, could be used as a counterselectablemarker. We now report that we have developed a counterselectablemarker by using glnQ with GGH selection and have used this systemto isolate mutant GBS strains that have the chromosomal capsularpolysaccharide regulatory gene cpsB replaced by the chloramphenicolacetyltransferase (cat) gene.

Selection against glnQ-expressing GBS by GGH is robust

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Selection against glnq...

We previously isolated COH1-GT1, a Tn917 transposon mutant derivativeof a virulent GBS strain (COH1). COH1-GT1 is deficient in glutaminetransport due to a Tn917 insertion in glnQ, a homologue of agene from Escherichia coli that is required for high-affinity,energy-dependent glutamine transport (14). We previously demonstratedthat COH1-GT1 was resistant to GGH at 100 µg/ml when grownin a minimal medium containing M9 salts, 1% glucose, and 0.1%yeast extract (M9GYE), whereas the growth of COH1 was completelyinhibited. We have subsequently demonstrated similar findingswith inocula as high as 10⁵ CFU/plate (data not shown). Theseresults demonstrate that the glnQ-mediated sensitivity to GGHis sufficiently robust to allow for use of glnQ as a counterselectablemarker.

Expression of glnQ in trans confers sensitivity to GGH in a glnQ mutant GBS strain

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Expression of glnq in...

We hypothesized that the glnQ gene expressed on a plasmid couldbe used as a counterselectable marker when used in the backgroundof a host lacking the glnQ gene, such as COH1-GT1. To test thishypothesis, we used pAG200, a complementation plasmid we hadpreviously constructed which contains the glnQ gene expressedin the gram-positive shuttle vector pDC123. pAG200 is able tocomplement the glutamine transport defect of COH1-GT1 (14).We plated COH1-GT1 containing pAG200 onto M9GYE agar with 100µg of GGH and 10 µg of chloramphenicol/ml to maintainthe presence of the plasmid. The results are shown in Fig. 1.We confirmed that COH1-GT1 is able to grow in the presence ofGGH (Fig. 1A). COH1-GT1 with pAG200 was able to grow on mediumcontaining chloramphenicol (Fig. 1B) but not on medium containingboth chloramphenicol and GGH (Fig. 1C). These results demonstratedthat pAG200 confers sensitivity to GGH in a ${Delta}$ glnQ backgroundand thus acts as a counterselectable marker.

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FIG. 1. pAG200 confers sensitivity to GGH in the background of COH1-GT1. (A) COH1-GT1, with GGH; (B) COH1-GT1:pAG200, with chloramphenicol; (C) COH1-GT1:pAG200, with chloramphenicol and GGH.

Creation of derivatives of GBS strains COH31rs and A909 with mutations in glnQ

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Creation of derivatives of...

We then sought to create ${Delta}$ glnQ host strains from laboratory strainsof GBS with high transformation efficiencies. Because COH1 hasa very low transformation efficiency ( $~$ 10³ CFU/µg of DNA),much of the genetic manipulation of GBS has been performed onlaboratory strains such as A909 and COH31rs that have much highertransformation efficiencies ( $~$ 10⁵ CFU/µg of DNA). We thereforeundertook to create derivatives of these laboratory strainswith site-directed mutations in glnQ that could then be usedas hosts for a glnQ counterselectable marker system. We createdsite-directed mutations in glnQ in A909 and COH31rs by usingpAG101, a plasmid containing the glnQ gene with an 80-bp internalfragment replaced with an erythromycin resistance gene (erm)in a TS background as previously described (14). The presenceof the mutations was confirmed by PCR as described earlier.One isolate for each host was designated A909-DLS1 and COH31-DLS1,respectively, and used as the GGH-resistant host in subsequentexperiments.

Generation of cpsB allelic exchange mutants

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Generation of cpsb allelic...

We then tested the utility of glnQ as a counterselectable markerin the creation of allelic exchange mutations in GBS by usingthe cpsB gene as a test case. cpsB is the second gene in thecapsular polysaccharide locus of GBS and appears to be involvedin regulation of capsule expression (4). A previously described3.0-kb EcoRI/EcoRV fragment from the capsule region of COH1containing the cpsB, along with adjacent fragments of the cpsAand cpsC (2), was cloned into pHY304, a TS shuttle vector encodingerythromycin resistance, to create pHY306. An exact allelicreplacement of the cpsB gene with the cat gene was then createdby using an in vivo ligation. A DNA fragment containing thecpsA and cpsC gene fragments flanking cpsB, together with vectorsequences, was created by PCR by using pHY306 as a templateand outward-reading primers (Table 1, cpsA3'R and cpsC5'F) thatwere synthesized with a 5' extension consisting of 20bp of the5' and 3' ends of cat, respectively. The cat gene was amplifiedby using pDC123 as a template, and primers cat F and cat R (Table1). Both PCR products were simultaneously electroporated intothe E. coli strain MC1061 and transformants were selected onLA with 10 µg of chloramphenicol/ml. The integrity ofone resulting clone was confirmed by limited restriction mapping,and designated pHY306.

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TABLE 1. Bacterial strains, plasmids, and primers used in this study

A kanamycin resistance cassette and the glnQ gene were thenadded to create pDLS104. The kan gene from pCIV2 was amplifiedwith primers kanF and kanR containing NotI sites (Table 1),digested with NotI, and ligated into pBluescript II SK(+) (Stratagene,La Jolla, Calif.) to create pDLS101. The insert from pHY306was released with EcoRV and EcoRI, and cloned into pDLS101 digestedwith EcoRI and HincII to create pDLS102. The glnQ gene frompAG200 was then cloned into pDLS102 by using XhoI and KpnI tocreate pDLS103. The entire insert from pDLS103 containing glnQ,kan, and the allelic exchange construct from pHY306 was clonedinto pHY304 by using SacII and ClaI to create pDLS104.

To create allelic exchange mutations in cpsB, pDLS104 was transformedinto COH31-DLS1 and A909-DLS1. Transformed strains were grownovernight in Todd-Hewitt broth with 500 µg of kanamycin/mlat 30°C, diluted 1:100, and grown overnight at 37°Cin the absence of antibiotic selection to cure the plasmid.Approximately 10⁶ CFU were then subjected to GGH selection inthe presence of chloramphenicol on M9GYE. Individual colonieswere then replica plated onto Todd-Hewitt broth with 10 µgof chloramphenicol and 500 µg of kanamycin/ml. A totalof 31 of 42 (74%) of the COH31-DLS1 derivatives and 22 of 31(71%) of the A909-DLS1 derivatives gave the expected antibioticphenotype (chloramphenicol resistant, kanamycin sensitive),indicating that they had lost the plasmid kan marker but hadretained the cat gene.

A single clone in each background was then designated COH31-DLS2and A909-DLS2, respectively, and tested for the presence ofthe allelic exchange mutation by Southern blot analysis. Thecat gene probe was created by PCR by using pDC123 DNA as a templateand the catF and catR primers. The cpsB probe was created byPCR with genomic COH1 DNA as a template, and the cpsBF and cpsBRprimers. Both probes were labeled with digoxigenin by usingthe DIG Chem-Link labeling and detection set (Roche AppliedScience, Indianapolis, Ind.) with HindIII-digested chromosomalDNA from COH31-DLS2 and A909-DLS2 according to the manufacturer'sprotocol. The results are shown in Fig. 2. The cpsB region probe(left lanes) gave the expected 1.1-kb band for strains COH31rs(lane 1) and A909 (lane 3). No band hybridizing to the cpsBgene was seen for COH31-DLS2 (lane 2) or A909-DLS2 (lane 4),indicating that, as expected, the cpsB gene is not containedin these strains. A cat gene probe gave the expected 3.1-kbband for both COH31-DLS2 (lane 2) and A909-DLS2 (lane 4), confirmingthe presence of the expected allelic exchange mutation. Overall,these results demonstrate that an allelic exchange can be rapidlyand efficiently performed in GBS with GGH as a counterselectablemarker.

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FIG. 2. Southern blot analysis of cpsB and cat genes of allelic exchange mutants. Genomic GBS DNA was digested with HindIII, and Southern blot analysis was carried out as described with probes specific for cpsB (left lanes) and cat (right lanes). Lanes: 1, COH31rs DLS1; 2, COH31rs DLS2; 3, A909 DLS1; 4, A909 DLS2.

GGH counterselection could conceivably be used for a wide varietyof organisms. In theory, GGH counterselection could be usedwith any organism that requires an intact glutamine transportgene for sensitivity to GGH. This phenomenon has been describedfor the glnQ gene of Bacillus stearothermophilus (16), Rhodobactercapsulatus (18), Rhodobacter sphaeroides (6), and the GNP1 geneof Saccharomyces cerevisiae (19).

In addition, glnQ homologues are found in wide variety of organisms,and the feasibility of GGH counterselection can be easily ascertainedby comparing the sensitivity of wild-type and glnQ mutant strainsto GGH. Testing for GGH sensitivity is generally done undernitrogen-limiting conditions that result in the induction ofglutamine transport genes. Thus, one requirement for efficientGGH selection is the ability of the host organism to grow innitrogen-poor minimal medium. Thus, GGH selection may not beapplicable to bacteria that require complex media for growth.

A counterselectable marker system for gram-positive bacteriawith rpsL has been described for S. pneumoniae. This systemwas used to create gene replacements without introduction ofantibiotic resistance markers; we speculate that similar genereplacements could be introduced into GBS by using GGH counterselection.

One drawback of both the glnQ and rpsL systems is that boththe glnQ mutations we describe (14) and rpsL mutations in otherhosts (7, 13) can create fitness defects in vivo. Thus, mutantscreated by using these systems may not be optimal for use insome applications, such as virulence testing in vivo. Hydrazidesof several amino acids have been used to demonstrate that particulargenes are required for transport of particular amino acids.This suggests that other amino acid transport genes, togetherwith their cognate amino acid hydrazides, could be used to createcounterselectable marker systems. We speculate that such systemsmight not impose fitness constraints that complicate the interpretationof data from these mutants obtained by using animal models ofinfection.

ACKNOWLEDGMENTS

We thank Craig Rubens, Amanda Jones, and Anne Clancy for helpfulsuggestions.

This study was supported by National Institutes of Health grantR29 AI41484-01 to G.S.T.

FOOTNOTES

* Corresponding author. Mailing address: Division of Infectious Disease and Department of Pediatrics, Children's Hospital and Regional Medical Center and University of Washington, 4800 Sand Point Way N.E., Seattle, WA 98105. Phone: (206) 987-2073. Fax: (206) 987-7311. E-mail: gtamura{at}u.washington.edu.

REFERENCES

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