Identification of essential genes in Staphylococcus aureus using inducible antisense RNA

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[8] Identification of Essential Genes in S t a p h y l o c o c c u s aureus Using Inducible A n t i s e n s e RNA By YINDUO JI, GARY WOODNUTr, MARTIN ROSENBERG,

and MARTIN K. R. BURNHAM Introduction Conditional disruption of gene expression is an important approach for addressing information on genes essential for bacterial growth or pathogenesis. This information is particularly useful for validating molecular targets for antibiotic discovery and vaccine development. Antisense technology is an effective approach to down-regulate expression of specific genes. It has been used widely to interfere with eukaryotic gene expression through injection of synthetic oligonucleotides complementary to mRNA I and by the synthesis of antisense RNA from DNA cloned in an antisense orientation. 2 However, the antisense method has not been used routinely to inhibit gene expression in bacteria, even though there is evidence that antisense regulation occurs naturally in bacteria during plasmid, phage, and chromosomal replication. 3 Reports have demonstrated that antisense RNA can effectively down-regulate gene expression in various bacterial systems. 4,5 Combining an antisense strategy with a regulated expression system is useful in identifying and characterizing essential genes critical to bacterial growth in vitro and in vivo. Moreover, such a strategy offers a unique approach to the study of bacterial pathogenesis and definition of virulence factors. Regulated antisense RNA can be used to decrease the expression of known genes during different stages of infection. The TnlO-encoded tet repressor has been successfully used to regulate expression of specific genes in prokaryotic cells 6 and has also been employed in a xy[/tet chimerical promoter system which has been shown to be strongly inducible in Bacillus subtilis using subinhibitory concentrations of tetracycline] We I A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. Mello, Nature 391, 806 (1998). a D. S. Kemodle, R. K. R. Voladri, B. E. Menzies, C. C. Hager, and K. M. Edwards, Infect. Immun. 65, 179 (1997). 3 E. G. H. Wagner and R. W. Simons, Ann. Rev. Microbiol. 48, 717 (1994). 4 L. Good and E E. Nielsen, Nat. Biotechnol. 16, 355 (1998). 5 S. A. Walker and T. R. Klaenhammer, Appl. Environ. Microbiol. 66, 310 (2000). 6 M. Stieger, B. Wohlgesinger, M. Karnber, R. Lutz, and W. Keck, Gene 226, 243 (1999). 7 M. Geissendorfer and W. Hillen, Appl. Microbiol. BiotechnoL 33, 657 (1990).

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[8]

constructed a tet regulatory system in S. aureus 8"9 and initially cloned an antisense hla (or-hemolysin) fragment downstream of the inducible xyl/tet promoter-operator fusion to demonstrate that this tet regulatory system can function in S. aureus on induction of antisense to hla. It was found possible to down-regulate expression of the chromosomal hla in vitro and to eliminate virulence of S. aureus in an animal model of infection using anhydrotetracycline (ATc), a nonantibiotic analog of tetracycline, as inducer. 8 By creating a random library of inducible antisense clones we have been able to implement a strategy to find genes relevant to both viability and pathogenesis. C o n s t r u c t i o n of S t a p h y l o c o c c a l C h r o m o s o m a l DNA R a n d o m L i b r a r y In order to construct a more random antisense library the fragments of genomic DNA in the range of 200 op to 800 op are produced by mechanical shearing of chromosomal DNA of the clinically derived strain Staphylococcus aureus WCUH29 in a nebulizer. Forty/zg chromosomal DNA in 1 ml of 50% (v/v) glycerol, 0.3 M sodium acetate (pH 7.5) solution is added to the chilled nebulizer chamber, the nitrogen pressure adjusted to 40 psi, and the DNA solution nebulized for 30-60 sec at 4 ° and aliquoted into two 1.5-ml Eppendorf tubes containing 0.9 ml of 100% ethanol, then mixed and chilled at - 2 0 ° overnight. The nebulized DNA is precipitated by centrifugation at 14,000 rpm for 10 min and washed with 70% (v/v) ethanol, air-dried, and dissolved in 50 #l of I0 mM Tris-HCl (pH 8.5). Since mechanical shearing of DNA can result in broken ends or produce a mixture of 3'-OH, 5'-P termini and 3'-P, 5'-OH termini, the nebulized DNA is digested with Bal31 nuclease to convert all the ends to 3'-OH and 5t-P termini suitable for blunt-end ligation. Briefly, in the digestion solution 50/zg DNA fragments are incubated with 2.5 U Bal31 (New England Biolabs, Beverly, MA) in the reaction buffer at 30 ° for 5 min and 10/xl of 0.5 M EDTA is added into the reaction tube to stop further digestion. The Bal31-digested DNA fragments are electrophoresed in TAE buffer in 0.8% agarose gel containing 0.05 keg/ml of ethidium bromide. A slice of the gel covering 200 to 800 bp is excised and put into an Eppendorf tube. The DNA fragments are purified from the gel using QIAEXII gel extraction kit (Qiagen, Valencia, CA). Subsequently the sized DNA fragments are ligated into an inducible vector pYJ335, a plasmid carrying ampicillin and erythromycin resistance markers and able to replicate in Escherichia coli and S. aureus (Fig. IA). The ligated DNA is electroporated into 25 /zl of E. coli ElectroMax DHIOB Cells (Gibco-BRL, Gaithersburg, MD) in a 0.1 cm 8 y. Ji, A. Marra, M. Rosenberg, and G. Woodnutt, J. Bacteriol. 181, 6585 (1999). 9 k. Zhang, E Fan, L. M. Palmer, M. A. Lonetto, C. Petit, L. L. Voelker, A. S. John, B. Bonkosky, M. Rosenberg, and D. McDevitt, Gene 255, 297 (2000).

[8]

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ANTISENSE STRATEGY FOR GENE IDENTIFICATION

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FIG. 1. (A) Construction of the inducible antisense S. aureus library. S. aureus WCUH29 chromosomal DNA was sheared, treated with Bal31, sized in the range of 200 bp to 1000 bp, and ligated into the E c o R V site of downstream Pxyllteto in ATe inducible vector pYJ335 [for construction of this plasmid, see Y. Ji, A. Marra, M. Rosenberg, and G. Woodnutt, J. Bacteriol. 181, 6585 (1999)]. The ligation of DNA was electroporated into E. coli DH 10B using Amp as selection. Transformants were pooled and incubated in LB-Amp (100/zg/ml) for amplification and purification of library plasmid DNA, which was electroporated into S. aureus laboratory strain RN4220 and after passage into S. aureus clinical strain WCUH29. (B) Screening for essential genes. The electrotransformants, WCHU29 carrying library plasmid DNA, were selected on TSA-Erm (5/zg/ml) and duplicated onto TSA-Erm plates with ATe and without ATe. Growth-defective and conditional lethal colonies on TSA-Erm with ATe were selected after overnight incubation.

cuvette at 1.8 kV, 200 f2, and 25 #F using the Bio-Rad (Hercules, CA) Gene Pulser unit. Transformed cells are incubated in 900/zl of S.O.C. medium (Gibco-BRL) at 37 ° for 45 min and plated (25/zl) onto LB-agar plates (ampicillin 100/zg/ml). Colonies are picked for PCR (polymerase chain reaction) analysis of diversity of insertion by mixing a colony in 5 0 / 4 PCR supermixture (Gibco-BRL) using two plasmid-specific primers, tetRfor1399 (5'CAATACAATGTAGGCTGC 3') and catUrev (5' AGq"rCATTTGATATGCCTCC 3'). If the library appears representative, colonies are collected from 20 LB-agar-Amp plates containing 500 to 1000 cfu (colony-forming units)/plate and inoculated into 100 ml of LB-Amp medium and incubate overnight.

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Screening Growth Defect and Lethal Colonies electrocompetent cells are prepared by the following procedure. The strain is inoculated in 500 ml of TSB medium and incubated at 37 ° with shaking (190 rpm) till OD600nm 0.4~).5. The bacterial cells are harvested by centrifugation at 8000 rpm for 10 min at 4 ° and washed four times in ice-cold sterilized 0.5 M sucrose with 0.5, 0.25, 0.125, and 0.0625 times the original culture volume. Finally, the cells are resuspended with 2 ml of sterilized 10% glycerol and aliquoted into 1.5-ml Eppendoff tubes (50 #l/tube) and stored in a - 8 0 ° freezer. Plasmid library DNA prepared from the collection of E. coli clones is subsequently electroporated into 50 #1 of S. a u r e u s laboratory strain RN4220 competent cells at 1.8 kV, 100 fl resistance, and 25/zF capacitance; then the electrotransformants are spread for single colonies on TSA-Erm (5/zg/ml) plates. The colonies are collected and inoculated into 50 ml TBS-Erm and incubated with shaking at 37 ° overnight. Plasmid library DNA is purified from this culture using a QIAprep Miniprep Kit (Qiagen) and electroporated into S. a u r e u s WCUH29 since this strain cannot accept foreign DNA directly from E. c o l i J ° To screen for lethal and growth defect events, the colonies are duplicated onto TSA-Erm plates either with inducer or without inducer (Fig. 1B). Colonies which grow normally on TSA-Erm without ATc, but which are missing or grow poorly on the replica TSA-Erm plates containing ATc after overnight incubation, will be evident. We have found that direct electroporation of the plasmid library isolated from RN4220 into WCUH29 could produce a more random library in WCUH29 (about 71% of the lethal and growth defective events represent different unique genes) in comparison with transducfion using ap11 (only 4.5% of lethal and deficient events represent different genes). The colonies displaying defective growth upon induction are isolated from the TSA-Erm plates and retested by streaking part of each colony onto the TSAErm-ATc and onto TSA-Erm after incubation overnight. To further confirm the phenotype, plasmid DNA is purified from each strain and electroporated back into S. a u r e u s WCUH29 competent ceils and selected on TSA-Erm plates. After growth, colonies on the plate are replicated onto TSA-Erm containing different concentrations (100 to 1000 ng/ml) of ATc. In these experiments S. a u r e u s WCUH29 carrying the parent vector pY1335 (i.e., strain YJ335) grow normally on TSA-Erm at the different concentrations of ATc. In contrast, the S. a u r e u s strains carrying the transformed plasmids do not grow or grow poorly with different levels of ATc induction. S. a u r e u s

S. a u r e u s

DNA S e q u e n c i n g a n d B i o i n f o r m a t i c A n a l y s i s To determine the orientation and to identify the specific DNA fragments leading to lethal or growth defective events after induction, the DNA fragments are obtained i0 R. P. Novick,"MolecularBiologyof the Staphylococci."VCH Publishers,New York, 1990.

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by PCR amplification and sequenced using plasmid-specific primers tetRfor1399 and catUrev. The DNA sequence data is analyzed by using BLAST Homology Search against all bacterial genomes database developed by GlaxoSmithKline Pharmaceuticals Research and Development. An antisense orientation to the cloned fragment in the recombinant plasmid is indicated if the direction of the query sequence using catUrev primer is the same as the direction of subject amino acid sequence in the protein identified. Only 33% of the S. aureus strains displaying growth deficient or lethal phenotype when induced with ATc carry DNA fragments in the antisense orientation in the repression vector. Of these, 33% have chimeric or rearranged fragments at the cloning site (see detailed results in Ref. 11 Q u a n t i t a t i v e T i t r a t i o n of E s s e n t i a l G e n e s in Vitro The ability to titrate down a gene product in vitro provides a powerful approach for quantitative analysis of gene essentiality. To characterize the titration of essential genes in vitro, the growth of S. aureus strains containing different essential antisense RNA constructs is determined by incubation of bacteria with different doses of ATc. The density of cells is determined at OD600nm after overnight incubation at 37 ° or by using kinetic assay. Dose-dependent inhibition of growth is observed during induction with various concentrations of inducer for S. aureus antisense mutant strains carrying essential antisense fragments. In contrast, the control strain S. aureus carrying pYJ335 does not show any obvious difference of growth in the presence of inducer.11 Q u a n t i t a t i v e T i t r a t i o n of E s s e n t i a l G e n e s in Vivo Because the tetracyclines are available in many body compartments after oral dosing, this regulated antisense system provides a unique tool for determining essentiality of a gene during infection and for studying pathogenesis in this organism. To titrate the expression level of expression of essential genes in vivo, we have chosen a murine model of hematogenous pyelonephritis as it results in a localized kidney infection from which bacteria are readily recovered.8 Five mice per group are infected with about 107 cfu of bacteria via an intravenous injection of 0.2 ml of bacterial suspension into the tail vein using a tuberculin syringe. Different doses of ATc are given orally in 0.2-ml doses (containing 5 ttg/gram weight of Erm) to infected mice on days 1, 2, and 3 after infection. The mice are sacrificed by carbon dioxide overdose 2 hr after the last dose of Tc induction. Kidneys are aseptically removed and homogenized in 1 ml of PBS for enumeration of viable bacteria. As a control, approximately 5 log cfu of Y1335 should be recovered from infected kidneys at day 3 either in the presence or absence of ATc induction. Similarly, about 5 log cfu of antisense mutants which are wild-type S. aureus carrying l I y. li, B. Zhang, P. Warren, G. Woodnutt, M. Burnham, and M. Rosenberg, Science 293, 2266 (2001).

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essential gene antisense constructs should be recovered from infected kidneys in the absence of induction. In contrast, no bacteria or less than 1 log cfu bacteria should be recovered from infected kidneys following induction of antisense using 0.5#g/gram mouse of ATc. u Also, the effect of essential antisense RNA induction on the survival of bacteria should be ATc-dose dependent. On some occasions induction of antisense in vivo does not lead to bacterial clearance although in vitro induction gives rise to cessation of growth. This suggests to us that interference of expression of the gene concerned in vivo has consequences that are not bactericidal.

[9] Transposomes: A System for Identifying Genes Involved in Bacterial Pathogenesis By LES M. HOFFMAN and JERRY J. JENDRISAK

Transposome

Formation from Tn5 Transposable Elements

Transposons are DNA elements that can move from one genetic location to another. Bacterial transposons of the Tn class are used extensively as research tools in molecular biology. They contain defined terminal inverted repeats and encode a transposase that excises the element from a donor site and rejoins it to DNA at a second location. The molecular details of Tn5 transposition have been well characterized owing to its single subunit transposase and to the development of an in vitro transposition system.1 Tn5 normally has a very low rate of transposition in bacteria. A hyperactive triple mutation of the Tn5 transposase was created which facilitates in vitro transposition studies. 1 When hyperactive Tn5 transposase was combined with a transposon with inverted repeat ends containing a mosaic of outer end (OE) and inner end (IE) sequences, 2 in vitro transposition efficiency was significantly improved. The mosaic ends (MEs) are 19 base pairs (bp) long and are the only sequences required for transposase recognition. Any sequence between ME repeats can be mobilized by transposition. During the excision of the transposable element from the donor DNA, a synaptic complex is formed in which the transposon ends are brought together by the dimerization of the transposase. Transposition intermediates called "transposomes" are complexes between two transposase molecules and any DNA with ME inverted repeat ends. 3 Transposomes l I. Y. Goryshin and W. S. Reznikoff, J. Biol. Chem. 273, 7367 (1998). 2 M. Zhou, A. Bhasin, and W. R. Reznikoff, J. Mol. Biol. 276, 913 (1998). 3 I. Y. Goryshin, J. Jenddsak, L. M. Hoffman, R. Meis, and W. S. Reznikoff, Nat. Biotechnol. 18, 97

(2000).

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