Multiple Chromosomes in Bacteria: The Yin and Yang of trp Gene Localization in Rhodobacter sphaeroides 2.4.1

Corresponding author: Samuel Kaplan, Department of Microbiology and Molecular Genetics, University of Texas Medical School, 6431 Fannin St., Houston, TX 77030., skaplan{at}utmmg.med.uth.tmc.edu (E-mail)

Communicating editor: R. MAURER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The existence of multiple chromosomes in bacteria has been known for some time. Yet the extent of functional solidarity between different chromosomes remains unknown. To examine this question, we have surveyed the well-described genes of the tryptophan biosynthetic pathway in the multichromosomal photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1. The genome of this organism was mutagenized using Tn5, and strains that were auxotrophic for tryptophan (Trp^-) were isolated. Pulsed-field gel mapping indicated that Tn5 insertions in both the large (3 Mb CI) and the small (0.9 Mb CII) chromosomes created a Trp^- phenotype. Sequencing the DNA flanking the sites of the Tn5 insertions indicated that the genes trpE-yibQ-trpGDC were at a locus on CI, while genes trpF-aroR-trpB were at locus on CII. Unexpectedly, trpA was not found downstream of trpB. Instead, it was placed on the CI physical map at a locus 1.23 Mb away from trpE-yibQ-trpGDC. To relate the context of the R. sphaeroides trp genes to those of other bacteria, the DNA regions surrounding the trp genes on both chromosomes were sequenced. Of particular significance was the finding that rpsA1, which encodes ribosomal protein S1, and cmkA, which encodes cytidylate monophosphate kinase, were on CII. These genes are considered essential for translation and chromosome replication, respectively. Southern blotting suggested that the trp genes and rpsA1 exist in single copy within the genome. To date, this topological organization of the trp "operon" is unique within a bacterial genome. When taken with the finding that CII encodes essential housekeeping functions, the overall impression is one of close regulatory and functional integration between these chromosomes.

TEN years ago, the first description of a bacterium possessing multiple chromosomes was published (SUWANTO and KAPLAN 1989A , SUWANTO and KAPLAN 1989B ). This organism, Rhodobacter sphaeroides 2.4.1, was shown to possess two circular chromosomes of 3.0 (CI) and 0.9 (CII) Mb in size. Until that time, the dogma that bacteria always had one circular chromosome went unquestioned.

R. sphaeroides is a photosynthetic member of the -3 group of Proteobacteria (WOESE et al. 1990 ). Other members of this group, i.e., Agrobacterium tumefaciens (ALLARDET-SERVENT et al. 1993 ), Brucella melitensis (MICHAUX et al. 1993 ), Paracoccus denitrificans (WINTERSTEIN and LUDWIG 1998 ), and Ochrobactrum anthropi (JUMAS-BILAK et al. 1998 ) have also been shown to possess multiple chromosomes. However, bacteria possessing multiple chromosomes are not unique to the -3 group. It has also been shown that Leptospira interrogans (BARIL et al. 1992 ), Burkholderia cepacia (RODLEY et al. 1995 ), and more recently Vibrio cholerae (TRUCKSIS et al. 1998 ), members of the Spirochaetales ß- and -Proteobacteria, respectively, also possess multiple chromosomes. Therefore, a decade after the initial discovery, the existence of multiple chromosomes in bacteria is known to be widespread. What remains unclear is the evolutionary selection for this genomic architecture and its biological significance.

We have examined a number of genes of R. sphaeroides and have found that some genes occur in multiple copies that are distributed between the two chromosomes. These include three rRNA operons (rrnA, rrnB, and rrnC), each encoding in the following order: 16S rRNA, tRNA^Ile, tRNA^Ala, 23S rRNA, 5S rRNA, and tRNA^Fmet. One of these rRNA operons, rrnA, is found on CI, whereas rrnB and rrnC are found on CII (DRYDEN and KAPLAN 1990 ). Other genes, including hemA/hemT (5-aminolevulinic acid synthase, NEIDLE and KAPLAN 1993 ), rdxA/rdxB (redox sensors, NEIDLE and KAPLAN 1992 ), rpoN_I/rpoN_II (sigma factors), groEL_I/groEL_II (chaperones) and many genes encoding the enzymes of the reductive Calvin cycle pathway cbbA_I/cbbA_II (HALLENBECK et al. 1990B ; TABITA et al. 1992 ), and cbbP_I/cbbP_II (HALLENBECK et al. 1990A ; TABITA et al. 1992 ) have also been shown to be duplicated between CI and CII, respectively. The isozymes encoded by these duplicate genes are often structurally similar, but in most cases have been shown to be differentially regulated.

Sequence sampling of CII-specific cosmids has revealed database matches to several hundred known genes (CHOUDHARY et al. 1997 , CHOUDHARY et al. 1999 ). This suggests that CII is in many respects like any other bacterial chromosome. It was also shown that many sequences present on CII do not return database matches. This suggests that many functions apparently unique to this organism reside on CII. Therefore, CII is not a truncated copy of its larger CI "sib." This was verified when Tn5 insertions into CII were shown to result in different auxotrophic phenotypes (CHOUDHARY et al. 1994 ). This suggests that essential, nonduplicated housekeeping functions are encoded on this chromosome.

The synthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan, and a number of other aromatic compounds, initially share a common biosynthetic pathway that has been studied extensively (CRAWFORD 1989 ; NICHOLS 1996 ; PITTARD 1996 ). The tryptophan branch of the pathway begins by the action of anthranilate synthetase (encoded by trpD and trpE) on the substrates chorismate and L-glutamine. The subsequent and sequential actions of the products of the trpG, trpF, and trpC genes lead to the penultimate compound in the pathway, indole-3-glycerol phosphate (IGP). In the last step, IGP and L-serine are the substrates for tryptophan synthase. This enzyme is a heterotetramer (₂ß₂) in which the and ß subunits are encoded by the genes trpA and trpB, respectively. With the exception of Acinetobacter calcoaceticus (KISHAN and HILLEN 1990 ), the trpBA genes have been shown to be adjacent and usually cotranscribed in that order.

In this article, we demonstrate that the R. sphaeroides genes encoding the enzymes of the tryptophan pathway are distributed between the two chromosomes. The genes trpA and trpE-yibQ-trpGDC are at two distant loci on CI, while trpF-aroR-trpB are at a single locus on CII. The genes trpF and trpB are separated by a hypothetical gene that we have designated aroR. In addition to the genes of the tryptophan pathway, we also describe neighboring genes, including cmkA and rpsA1. In Escherichia coli these genes are essential for chromosome replication and translation, respectively. Southern hybridization suggests that there is a single copy of rpsA1 and it is located on CII, upstream of trpF-aroR-trpB. To date, this is a unique genomic arrangement for the trp "operon" and the first demonstration in bacteria of genes that encode a single biosynthetic pathway that is distributed between two chromosomes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Bacterial strains, cosmids, and plasmids:
Those used are listed in Table 1. Unless otherwise stated, the bacterial strains were grown as follows: R. sphaeroides 2.4.1 and derivative strains were grown at 30° in either Luria-Bertani (LB) medium or Sistrom's minimal medium A (SMM) supplemented where appropriate with antibiotics: streptomycin/spectinomycin (Sm/Sp) 50 µg/ml, potassium tellurite K₂TeO₃ (Te) 10 µg/ml, tetracycline (Tc) 1 µg/ml, and trimethoprim (Tp) 50 µg/ml. Media for the growth of auxotrophs were supplemented with 20 µg/ml L-tryptophan. E. coli strains were grown at 37° in LB medium supplemented where appropriate with antibiotics: ampicillin (Ap) 100 µg/ml, Tc 15 µg/ml, and Sm/Sp 50 µg/ml. E. coli strains DH5 and DH5phe^- were used for routine cloning. E. coli S17-1 was used for transferring mobilizable plasmids and cosmids to R. sphaeroides. Bacterial conjugation was carried out on LB plates (without antibiotics) at 30°.

Isolation of auxotrophs:
Bacterial conjugation was carried out as described previously (DONOHUE and KAPLAN 1992 ; CHOUDHARY et al. 1994 ). The mobilizable suicide plasmid pSUPTn5TpMCS that carries the transposon Tn5TpMCS (Tn5) was introduced into R. sphaeroides 2.4.1S by mating from E. coli S17-1. Matings were carried out, and exconjugants were plated on LB Te Tp plates. Colonies were replica plated to minimal SMM Te Tp plates. Colonies that were auxotrophic were purified and tested for their ability to grow without tryptophan. This led to the isolation of Trp^- strains CM01, CM02, CM03, CM05, and CM06.

The transposon used has three features relevant to this report: it carries a Tp-resistance (Tp^r) gene; it has a unique EcoRI site outside the Tp^r gene; and it has sites for the restriction enzymes AseI, DraI, SnaBI, and SpeI. These sites occur rarely in the R. sphaeroides genome (SUWANTO and KAPLAN 1989A , SUWANTO and KAPLAN 1989B ). Digestion of chromosomal DNA from Tn5 insertion strains (using these enzymes) followed by transverse alternating field electrophoresis (TAFE) permitted the site of Tn5 insertion to be determined.

Cloning the R. sphaeroides regions flanking Tn5 insertions:
The DNA flanking the sites of transposon insertion was cloned as described previously in detail (MACKENZIE et al. 1995 ). This method was used to generate plasmids pCM01, pCM02, pCM03, pCM04, pCM05, and pCM06. In the case of plasmid pCM02Sal, the enzyme SalI (there are no sites for this enzyme in the Tn5) was used to subclone the intact transposon with flanking R. sphaeroides DNA.

Mapping sites of Tn5 insertion by TAFE gel electrophoresis:
DNA plugs were prepared and then digested as described previously (MACKENZIE et al. 1995 ). Fragments were resolved on a 1x TBE and 1% SeaKem GTG gel (FMC, Rockland, ME) using a GeneLine II TAFE System. Electrophoresis was carried out at 10° using the following pulse conditions: stage 1, 30-sec pulse for 5 hr at 350 mA; stage 2, 45-sec pulse for 8 hr at 370 mA; stage 3, 60-sec pulse for 8 hr at 370 mA; stage 4, 90-sec pulse for 5 hr at 390 mA.

DNA sequencing of complete genes and chromosomal regions:
Plasmids pCM01, pCM02, pCM03, pCM04, pCM05, and pCM02Sal, as well as cosmids pUI8668 and pLX1P20, were used for further subcloning of the region surrounding trpEGDC for sequencing. Cosmid pUI8063 and pUI8536 were used in the same way for regions surrounding trpA and trpFB, respectively. Successive rounds of cloning and sequencing allowed the appropriate DNA fragments to be sequenced on both strands.

Sequencing reactions:
Plasmid DNA was prepared using Wizard Plus SV minipreps (Promega, Madison, WI). Sequencing of Tn5-R.sphaeroides hybrid fragments used three primers, GW25 (5'-TTCAGGACGCTACTTGTGTA-3'), which is complementary to the IS50 of the transposon, and pBluescript T3 and T7 primers. GW25 was used to sequence from the transposon into the flanking R. sphaeroides DNA. All other plasmid-sequencing reactions used the T3 and T7 primers alone. PCR products were sequenced using specific PCR primers (Table 2). DNA sequencing was performed at the Microbiology and Molecular Genetics Core Facility using Big-Dye chemistry and an ABI 377A sequencer (Applied Biosystems, Foster City, CA).

PCR:
Reactions had the following components: 200 µM dNTPs, 25 pmol of each primer (Table 2), 5% v/v DMSO, 10 ng of template DNA, and 2.5 units of Pfu DNA polymerase (Stratagene, La Jolla, CA). Cycling times were as follows: step 1, 95°, 1 min; step 2, 15° below lowest primer Tm, 1 min; step 3, 72°, 2 min; step 4, 24 times to step 1; step 5, 72° for 4 min in a PTC-100 thermal cycler (M.J. Research, Watertown, MA). PCR primer pairs were designed so that the PCR products were from the internal regions of the genes described. The primer pairs are listed in Table 2.

Cosmid libraries:
A previously described and ordered CII-specific pLA2917 cosmid library was available (CHOUDHARY et al. 1994 ). A second library was constructed using the cosmid vector pLAFRx, as described by SAMBROOK et al. 1989 . After in vitro packaging with Gigapack Gold III (Stratagene, La Jolla, CA), the cosmids were introduced into E. coli XL1-Blue MRA. The cosmids were pronged onto plates, grown overnight, and then screened for colony hybridization as described.

Complementation:
Recombinant cosmids pUI8063 and pUI8536 and the vector pLA2917 were introduced into E. coli S17-1. They were then mated to an R. sphaeroides trpA mutation (CM09 x pUI8063; CM09 x pLA2917) and trpB^- mutation (CM06 x pUI8536; CM06 x pLA2917) on LB plates as described previously (CHOUDHARY et al. 1994 ). The R. sphaeroides strains were selected initially on LB Tc Te plates. Twenty Tc^r colonies (from four independent crosses) were then tested for their ability to grow on SMM, SMM Tc, and SMM Tc Sm/Sp (::trpA^-) or SMM Tc Tp (Tn5::trpB^-) plates. Complemented strains could grow on all three plates, i.e., CM10 and CM07.

Southern blotting:
Gels were depurinated and then transferred by alkali to Hybond N⁺ membranes (Amersham, Piscataway, NJ) using standard techniques (SAMBROOK et al. 1989 ). Probes were radiolabeled using [-³²P]dCTP and a RadPrime DNA labeling system (GIBCO-BRL, Gaithersburg, MD). Probes were purified using a Sephadex G50 spin column and then denatured before use.

Hybridization:
Southern blots and colony lifts were hybridized using standard techniques (SAMBROOK et al. 1989 ). Hybridization and washing were carried out at 55° or 65° for blots and lifts, respectively. Washing was carried out at the hybridization temperature in the following solutions: 4x SSC and 0.1% SDS for 10 min done twice, 1x SSC and 0.1% SDS for 10 min done twice. For lifts, an additional wash in 0.1x SSC and 0.1% SDS for 10 min was done twice.

Cloning trpA:
Primers were made to the R. capsulatus trpA sequence (Table 2), and PCR was performed as described above. The PCR product was gel purified and then sequenced. The DNA sequence matched other trpA genes. The PCR product was then used to probe the pLA2917 genomic library. Positively hybridizing cosmids were isolated and probed by Southern hybridization. A positively hybridizing 6.5-kb BamHI fragment was subcloned from cosmid pUI8063 into the BamHI site of pBS to give plasmid pCM07A. A 1.3-kb SmaI fragment containing trpA from pCM07A was subcloned into the EcoRV site of pBS to give pCM08A.

Gene disruptions:
The following strategy was used to disrupt the genes trpA, trpE, trpG, and aroR (Table 3). An omega () Sm/Sp cartridge carrying transcriptional terminators was used to make polar gene disruptions. The cartridge, carried on a SmaI fragment, was cloned into the gene of interest. The disrupted gene was then subcloned into the mobilizable suicide vector pSUP203. This construct was mated into R. spheroides 2.4.1. Exconjugants were selected on LB Sm/Sp tryptophan plates. They were then tested for their ability to grow without tryptophan.

View this table: [in this window] [in a new window]   Table 3. Sites of Tn5 or Sm/Sp insertion

trpA:
Plasmid pCM08A was linearized using StyI, which cuts within trpA at codon 164. The ends of the linearized plasmid were filled using Klenow fragment and then the Sm/Sp cartridge inserted, giving plasmid pCM09A. This plasmid was digested with PvuII, and a 3.8-kb fragment containing the interrupted trpA was inserted into ScaI-digested pSUP203 to give pCM10A. This plasmid was introduced into R. sphaeroides from E. coli S17-1, resulting in R. sphaeroides trpA mutation strain CM09.

trpE:
A 2.9-kb PstI fragment containing trpE from pCM03 was cloned into the PstI site of pBSII to give plasmid pCM11E. A 1.4-kb HindIII/HincII fragment containing trpE was excised from this plasmid and subcloned into the HindIII/HincII sites of pBSII to give pCM12E. An Sm/Sp cartridge was inserted into the BstEII site in trpE (between codons 171 and 172) to give plasmid pCM13E. A PvuII fragment from pCM13E was excised and inserted into the ScaI site of pSUP203, resulting in plasmid pCM14E. This plasmid was introduced into R. sphaeroides from E. coli S17-1, resulting in R. sphaeroides strain CM11.

trpG:
A 2.3-kb PstI fragment containing trpG from pCM03 was cloned into the PstI site of pBSII to give plasmid pCM15G. An Sm/Sp cartridge was inserted into the NsiI site within trpG (disruption of codon 43) to give plasmid pCM16G. A PvuII fragment containing the disrupted trpG was then inserted into the ScaI site of pSUP203 to give plasmid pCM17G. This plasmid was introduced into R. sphaeroides from E. coli S17-1, resulting in R. sphaeroides strain CM12.

aroR:
Cosmid pUI8536 was digested with BglII. A 1.3-kb fragment containing aroR was inserted into the BamHI site of pBS to give pCM18R. This plasmid was partially digested with SmaI. The Sm/Sp cartridge was then inserted. A plasmid containing the insertion at aroR codon 47 was selected (pCM19R). This was digested with PvuII, and a 3.8-kb fragment was taken and cloned into the ScaI site of pSUP203 to give plasmid pCM20R. This plasmid was introduced into R. sphaeroides from E. coli S17-1, resulting in R. sphaeroides aroR mutation strain CM08.

Sequence analysis:
DNA editing was carried out using Seqed (Applied Biosystems). Fragments were then assembled using Gelassemble (version 9.1, Genetics Computer Group, Madison, WI). PCR primers were designed using Primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi). BLASTX and BLASTP were used for database comparison through the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/).

Nucleotide accession numbers:
The sequences described in RESULTS have the following GenBank accession numbers: AF10704, trpA; AF107095, guaB, lctD, mosC; AF107096, hypothetical GTP-binding protein; AF108766, asmA [partial coding sequence (cds)], ybaU, trpE, yibQ, trpG, trpD, trpC, moaC, lexA, comE, gluS, cisY (partial cds); AF107093, cmkA, rpsA1, hipB, trpF, aroR, trpB, Synechocystis orf.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Screening for auxotrophs:
Using Tn5 mutagenesis of R. sphaeroides 2.4.1S, we recovered 33 auxotrophic strains. Five of these strains, CM01, CM02, CM03, CM05, and CM06, were auxotrophic for tryptophan (MACKENZIE et al. 1995 ). Strains CM03 and CM05 were capable of growth on minimal medium; however, they took 7–8 days to form colonies compared to 3–4 days for the wild type. The addition of tryptophan to their media restored their growth to wild-type rates. The other four strains did not show any visible growth unless tryptophan was added to the minimal media.

Placing Tn5 insertions on the physical map:
Digestion of R. sphaeroides strain 2.4.1S with the enzyme SpeI yielded a CI fragment of 735 kb. Digestion of strains CM01, CM02, CM03, and CM05 with SpeI resulted in the loss of this fragment. In its place, two fragments, each ~365 kb in size, were generated (Fig 1A). This indicated that the site of Tn5 insertion in these strains was on CI, within the central region of the 735-kb SpeI fragment. These strains showed resolvable restriction pattern differences, indicating that they were the result of independent transposition events. Further mapping studies (not shown) have localized their position to that shown on the physical map (Fig 1A). Because of their proximity, we have marked their position as a single chromosomal location in the figure.

View larger version (61K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Mapping the sites of Tn5 insertion. Tn5 insertion strains were made in a 2.4.1S background (see MATERIALS AND METHODS). Both panels show a TAFE gel containing DNA from R. sphaeroides 2.4.1S and Tn5 insertion strains that are Trp-. The leftmost lane in each gel contains a 50-kb marker ladder of concatenated genomes. The lowest rung of the ladder is 50 kb. Above each gel panel is the physical map of CI (left) and CII (right). Each physical map is composed of four concentric circles marked with restriction sites (moving from outer to inner concentric) for enzymes AseI, DraI, SnaBI, and SpeI. The zero position (0) is shown at 12 o'clock within each map. The distance markers shown around the inner circle are in increments of 500 kb (CI) and 100 kb (CII). The black arc represents the restriction fragment that is being examined in the gel below. The full arc is what is found in 2.4.1S. The two smaller arcs (resulting from cleavage of the full arc due to Tn5 insertion) represent the new DNA fragments found in the Trp- strains. The site of Tn5 insertion is shown as a lollipop. It has been placed in this position by using other enzymes for digestion (not shown). (A) R. sphaeroides 2.4.1S and CI Tn5 insertion strains CM03 (ybaU-), CM05 (ybaU-), CM01 (trpE-), and CM02 (trpD-) are shown. The DNA has been digested with SpeI. In 2.4.1S, an SpeI band of 735 kb is visible. In the Trp- strains, this band is absent (indicating its disruption by Tn5); instead, there is a 365-kb "doublet" that is not visible in the 2.4.1S strain. This indicates that the Tn5 insertion lies within the 735-kb CI SpeI fragment in these Trp- strains. It can be seen that the doublet bands get closer together. This indicates that the Tn5 insertions in YbaU are farther out from the center of the 735-kb SpeI fragment than those in trpE and trpD. These differences in fragment size, though visible, were not sufficient to be estimated on this gel. (B) R. sphaeroides 2.4.1S and CII Tn5 insertion strain CM06 (trpB-) is shown. The DNA has been digested with SnaBI. In 2.4.1S, a SnaBI band of 784 kb is visible. In the Trp- strain, this band is absent (indicating its disruption by Tn5); instead, there are two bands of 750 and 34 kb that are not visible in the 2.4.1S strain. This indicates that the Tn5 insertion lies within the 750-kb CII SnaBI fragment in the Trp- strain.

Digestion of the DNA of strain 2.4.1S with the enzyme SnaBI yields a CII fragment of 784 kb (Fig 1B). Digestion of the DNA of strain CM06 with this enzyme resulted in the loss of this fragment, which was replaced by two restriction fragments of 34 and 750 kb in size. This indicated that the site of this insertion was on CII. Further mapping studies (not shown) placed the Tn5 insertion at the position shown on the physical map (Fig 1B).

Cloning and sequencing the DNA flanking the site of Tn5 insertion:
We obtained Tp^r EcoRI subclones used for cloning and sequencing of each of the auxotrophic strains and used the strategy described in MATERIALS AND METHODS. The use of the sequencing primer GW25 revealed that in CI insertion strains CM01 and CM02, the transposon was located in the genes trpE and trpD (Table 4, Fig 2, and Table 5), which encode the enzymes anthranilate synthase (component I) and anthranilate phosphoribosyltransferase, respectively. The use of primer GW25 to sequence pCM06 revealed that in CII insertion strain CM06, the transposon was located within trpB (Fig 2 and Table 4), which encodes the ß-subunit of tryptophan synthase. These results also suggested that in R. sphaeroides, the genes for tryptophan biosynthesis were distributed between chromosomes CI and CII. The use of primer GW25 indicated that CI insertion strains CM03 and CM05 had Tn5 insertions in the gene ybaU, and that their insertions lay in opposite orientations within the gene (Fig 2 and Table 4). In E. coli, this gene encodes a peptidyl-prolyl cis-trans-isomerase, which assists in protein folding. It had been noted that both strains CM03 and CM05 grew very slowly in the absence of tryptophan. This result suggested that transposon insertions in the gene ybaU may be polar on the nearby trp genes. The possibility that YbaU is a regulator of trp gene expression or is required for the folding of their gene products has not been excluded.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Overview of gene organization. The three regions encoding the trp genes and their flanking sequences have been placed on the physical maps of CI and CII. Genes are shown as arrows indicating the direction of transcription. The trp genes are shown as black arrows, fully sequenced genes as gray arrows, and partially sequenced genes as white arrows. The sizes of the partially sequenced genes have been estimated from the size of the genes in the database that they matched. The location of Tn5 insertions and Sm/Sp cartridge insertions are indicated by inverted triangles and symbols, respectively. The exact locations of these insertions are provided in Table 3. Adjacent to the insertion is the name of the strain in Table 1 that carries that insertion. Beneath the genes are horizontal lines representing the R. sphaeroides-Tn5 EcoRI hybrid fragments with the names of the plasmids that carry them (Table 1). At the end of these fragments are the numbers 3, 7, or 25. These indicate the primers T3, T7, and GW25, which were used for sequencing the ends that gave database matches (Table 4). Inserts from plasmids pCM02 and pCM05 are split between the two levels of the CI "Christmas tree." All distances are shown approximately to scale; a scale bar is provided. Note that the Tn5 insertion strain CM04 (on the upper left branch of the tree) is an arginine auxotroph (Arg-). We looked briefly at this strain, which contained a Tn5 insertion that mapped 6 kb upstream of trpE. The Tn5-R.sphaeroides hybrid fragment from this strain was subcloned (pCM04) and sequenced using T3, T7, and GW25 primers (Table 4). Additional mapping, subcloning, and sequencing were also carried out. We did not fully sequence this region, but we obtained BLASTX matches (Table 5) and sufficient data to map the region as shown.

Sequencing trp genes and surrounding regions on CI:
To complete the sequence of trpD and trpE on CI and to determine if other trp genes lay nearby, we sequenced further up- and downstream from the sites of transposon insertion. DNA sequencing also located the precise site of the transposon insertions within the genes (see Table 3). Templates were obtained by subcloning from the R. sphaeroides-Tn5 hybrid EcoRI fragments. A DNA region of 14,548 bp was sequenced, and within it were found the trp genes, trpC and trpG, which encode the enzymes indole-3-glycerol phosphate synthase and anthranilate synthase (component II), respectively. This region contained 13 genes, and 11 of these (including trpE, G, D, and C) were sequenced to completion (Table 6). BLASTP searching of the database showed that the predicted translations of trpE, G, D, and C had high sequence identity to their counterparts in other organisms. The physical map positions of these genes are shown in Fig 2.

The DNA region between trpE and trpG did not give a clear match to any database sequence (Fig 2). Computer predictions suggested that a hypothetical gene within this region would encode a protein of 266 amino acid residues. We have called this hypothetical gene yibQ, as its predicted translation showed the closest relevant match to YibQ, a hypothetical protein from Haemophilus influenzae. This ORF is encoded on the opposite DNA strand to the trp genes, raising the possibility that trpE and trpG may have their own promoters.

In addition to genes for tryptophan biosynthesis, sequencing of this region revealed a number of other genes (Fig 2). Immediately downstream of trpC were two putative genes, moaC and moeA. It has been suggested that MoeA activates molybdenum by conversion to thiomolybdenum (HASONA et al. 1998 ). Both moaC and moeA are required for molybdopterin synthesis, an essential component of Mo-cofactor-containing enzymes, such as nitrate reductase. In the nodulating bacterium Bradyrhizobium japonicum, an operon having the same gene order (trpD, trpC, and moaC-like gene) was found to be essential for plant symbiosis (KUYKENDALL and HUNTER 1997 ).

Downstream of these genes lay a putative lexA/dinR gene. In E. coli, lexA encodes a repressor of the SOS genes, such as recA and uvrABC (DNA damage repair genes). BLASTP analysis indicated that the R. sphaeroides gene product showed greater homology to proteins from Gram-positive (DinR) than Gram-negative (LexA) bacteria. Previous work suggested that a RecA^- strain of R. sphaeroides was less sensitive to ultraviolet light than a RecA^- E. coli strain. The differences in sensitivity could not be explained in terms of G + C% composition or target size (CALERO et al. 1994 ; MACKENZIE et al. 1995 ). This new result adds to the possibility that the DNA repair mechanisms in R. sphaeroides could be regulated more like those in Gram-positive bacteria, such as Bacillus subtilis.

DNA sequences downstream of lexA showed matches to glutamyl-tRNA synthetases (encoded by gltX) and citrate synthases (cisY partial gene), the latter being a key enzyme in the TCA cycle. The region between lexA and gltX (and encoded by the opposite DNA strand) gave matches to several ComE proteins. These proteins, encoded by comE genes, are involved in competence and DNA uptake in Gram-positive bacteria; however, there is no evidence to suggest that R. sphaeroides is naturally competent.

Introducing Sm/Sp insertions into trpE and trpG:
To test the hypothesis that trpG was a functional gene, trpG was disrupted using an Sm/Sp cartridge (CM12). As a control, we disrupted trpE in the same way (CM11). Insertions in trpE and trpG, which were confirmed by Southern blot analysis (not shown), conferred an auxotrophic phenotype (Trp^-). However, the possibility exists that both trpG and trpE are nonfunctional, and that insertions in these genes have a polar effect on the downstream functional genes trpDC. Given their high sequence homology to other genes in the data base, we consider this possibility unlikely. Southern hybridization data (discussed below) further quell this conclusion.

Sequencing trp genes and surrounding regions on CII:
The DNA region on CII-neighboring trpB was sequenced using subclones generated from cosmid pUI8536. This cosmid formed part of an ordered set of clones defining CII, and had been mapped independently to the CII site of the Tn5 insertion. The Trp^- CII insertion strain, CM06, was restored to prototrophy by complementation when this cosmid was introduced by mating (CM07). When the cosmid vector (pLA2917) was introduced into CM06, it remained Trp^-. This provided additional evidence that the Trp^- phenotype was the result of the Tn5 insertion on CII and not the result of a second mutation at a different chromosomal location.

A CII region of 6203 bp from cosmid pUI8536 was sequenced, and the DNA was found to encode in the order cmkA, rpsA1, hip, trpF, aroR, and trpB (Fig 2 and Table 6). The predicted translation of trpB indicated that it encoded 409 amino acid residues, and in strain CM06, the transposon insertion was at codon 91. This suggested that the Tn5 insertion in trpB on CII had resulted in a Trp^- phenotype. Downstream of trpB and in the opposite orientation, a region matching a Synechocystis open reading frame (ORF) of unknown function was found.

The CII region upstream of trpF encoded three additional genes. The most distal of these, cmkA (formerly mssA), encodes cytidine monophosphate kinase. In E. coli, this gene is considered essential and is required to maintain the normal rate of chromosomal replication. Downstream of cmkA, we found the gene rpsA1. In E. coli, this gene is essential for translation and encodes the largest protein component of the ribosome (KITAKAWA and ISONO 1982 ). It has also been shown (in E. coli) that cmkA is cotranscribed with rpsA1 as part of the rpsA operon (FRICKE et al. 1995 ). The gene hip (himD) lies downstream of rpsA1. This gene encodes the ß-subunit of the integration host factor (IHF, RICE et al. 1996 ). The -subunit of this protein is encoded by the gene himA and has been shown to map to the 1105-kb CI AseI fragment (P. SEN and S. KAPLAN, unpublished results). We have partially sequenced the region immediately upstream of cmkA (data not shown). The result suggested that the gene aroA, which encodes the enzyme 3-phosphoshikimate-1-carboxyvinyltransferase, is within this region. A similar gene organization (aroA-ycaL-cmkA-rpsA1-himD) has been found in the region 960217–964217 of the E. coli genome. However, in E. coli, this region is neither followed nor preceded by the genes for tryptophan biosynthesis.

The region between trpF and trpB was used in a BLASTX search of the database. The result suggested that this region encoded a regulator of the C-P lyase pathway. An Sm/Sp insertion within this gene (CM08) did not result in a Trp^- phenotype. This suggested that trpB is not transcribed from the trpF promoter. Rather, it has its own promoter and lies downstream of the insertion, i.e., within the 323-bp region preceding the trpB start codon. The function of the gene lying between trpF and trpB is unknown. We have named it aroR (aromatic amino acid regulator) to reflect its location and a plausible function.

The isolation, sequencing, mapping, and complementation of trpA on CI:
It had been expected that trpA would be downstream of trpB, as this is the gene organization in every member of the -3 group of Proteobacteria examined to date. As a result, we carried out PCR using R. sphaeroides genomic DNA as a template and primers designed to trpA of R. capsulatus. A 600-bp DNA product was generated. After DNA sequencing, it gave a BLASTX match to database TrpA proteins, which are the -subunits of tryptophan synthase. This PCR product was used as a probe to screen an R. sphaeroides cosmid library, to which five cosmid clones hybridized. Their DNA was purified, and they were probed in a Southern blot with the trpA PCR product (result not shown). The result showed that trpA was located on a 6.5-kb BamHI fragment. This fragment was subcloned from cosmid pUI8063, and the region sequenced. Within this region, a putative trpA gene and four other putative genes were found (see Fig 2 and Table 6).

An Sm/Sp cartridge was inserted into the cloned trpA gene (pCM10A). This gene interruption was introduced into the R. sphaeroides genome as described in MATERIALS AND METHODS. From five independent matings, 34 Sm^r/Sp^r strains were isolated. Five of these were found to be Tc^s. These 5 strains were also tryptophan auxotrophs (Trp^-), suggesting that in these strains, a chromosomal interruption of trpA had occurred by a double-crossover event. This hypothesis was validated by genomic Southern blot (not shown). One of these TrpA^- strains was designated CM09.

To further confirm that the observed Trp^- phenotype was a result of the disruption of the trpA gene, cosmid pUI8063 (trpA⁺ Tc^r) or the cosmid vector pLA2917 (Tc^r) was introduced by mating into each of the five R. sphaeroides TrpA^- strains. Twenty colonies from each mating were restreaked on SMM Tc and SMM Tc tryptophan plates. Colonies that had received the vector alone grew only on SMM Tc with tryptophan. Colonies that had received cosmid pUI8063 grew on all three plates (e.g., CM10). This suggested that the disruption of the trpA gene in the five mutants was responsible for the Trp^- phenotype.

The Sm/Sp cartridge carries recognition sites for the restriction enzymes AseI and DraI. Use of these sites allowed us to locate trpA on the physical map. We mapped the five Tc^s TrpA^- strains to the same location on the map by TAFE pulse-field gel electrophoresis. The mapping of one of these strains, CM09, is shown as an example in Fig 3. It can be seen that in the 2.4.1 wild-type strain DraI (Fig 3A) and AseI (Fig 3B), digestion generates CI fragments of 800 and 910 kb, respectively. In the TrpA^- mutant, these fragments are absent. They are replaced by two CI DraI fragments of 625 and 175 kb (Fig 3A) and two CI AseI fragments of 485 and 425 kb (Fig 3B). These results suggested that trpA and trpB were on different chromosomes, and that trpA was located on the physical map ~1.23 Mb (149° counterclockwise) from the other CI trp genes (Fig 2 and Fig 3).

View larger version (49K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Mapping trpA on CI. A disruption in the trpA gene was made using an Sm/Sp cartridge in an R. sphaeroides 2.4.1 background. This gave TrpA- strain CM09. Both gel panels show a TAFE gel containing DNA from R. sphaeroides 2.4.1 in the left-hand lane. The right-hand lanes contain DNA from strain CM09. Between the gel panels is the physical map of CI, which is composed of four concentric circles marked with restriction sites (moving from outer to inner concentric) for enzymes AseI, DraI, SnaBI, and SpeI. The zero position (0) is shown at 12 o'clock within the map. The distance markers shown around the inner circle are in increments of 500 kb. The black arcs represent the restriction fragments that are being examined in the gel. The two full arcs are what is found in strain 2.4.1. The four smaller arcs (resulting from cleavage of the full arc caused by Sm/Sp insertion) represent the fragments found in the trpA- strain. The site of Sm/Sp is shown by an symbol on the map. The sizes of the fragments described below were determined on additional gels that are not shown. (A) Digestion of 2.4.1 DNA with the restriction enzyme DraI gave a gel band of 800 kb. In the TrpA- strain, this band is absent and is replaced by two smaller bands of 175 and 625 kb. This indicates that the Sm/Sp insertion lies within the 800-kb DraI fragment. (B) Digestion of 2.4.1 DNA with the restriction enzyme AseI gives a gel band of 910 kb. In the trpA mutation strain, this band is absent and is replaced by two smaller bands of 485 and 425 kb. This indicates that the Sm/Sp insertion lies near the middle of the the 910-kb AseI fragment. The only position of insertion that will satisfy the results shown in A and B is that shown on the physical map.

Gene fusions overlapping stop-start codons and ribosome-binding sites:
It had been shown previously that a number of genes in the tryptophan pathway are fused. For example, in Rhz. meliloti and its relatives, trpE and trpG are fused to give trp(EG), resulting in a fusion of the - and ß-subunits of anthranilate synthase into a single polypeptide. Examination of the trp genes of R. sphaeroides indicated that such gene fusions had not evolved. Of note, however, were the numbers of neighboring genes that shared overlapping stop and start codons (Fig 4); i.e., trpG stop overlaps with trpD start, trpC stop overlaps with moaC start, moaC stop overlaps with moeA start, and trpF stop overlaps with aroR start. The genes trpC, moeA, and aroR had putative ribosome-binding sites upstream of their start codons; however, ribosome-binding sites were not found upstream of the genes trpG, trpD, and moaC. All other tryptophan-related genes, trpA, trpB, trpE, and ybaU, were found to have putative ribosome-binding sites upstream of their initiation codons.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Overlapping start and stop codons. The overlapping start and stop codons are shown centered on the second base (T) of the ATG or GTG start codons. The A bases of the TGA stop codons are marked with asterisks.

TAFE Southern blot hybridization with trp, rpsA1, and aroR probes:
Internal primer pairs (Table 2) were made to the following genes: rpsA1, trpF, aroR, trpB, trpA, trpC, trpD, trpE, and trpG, and were used for PCR. After sequencing had verified that the expected fragments had been generated, they were used to probe R. sphaeroides AseI TAFE Southern blots (Fig 5). The results suggested that trpA was located on CI within the 910-kb AseI fragment, and that trpC, trpD, trpE, and trpG were located on CI within the 1105-kb AseI fragment. Genes rpsA1, trpF, aroR, and trpB were found to be located on CII. A shorter exposure (not shown) indicated that these genes hybridized to the 360-kb CII AseI fragment rather than to the slightly smaller 340-kb CII AseI fragment.

View larger version (98K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Localization of genes to the physical map by hybridization. R. sphaeroides 2.4.1 DNA was digested with AseI. Ten lanes of a TAFE gel were loaded with equivalent amounts (one-eighth of a plug) of DNA. The gel was run and then photographed. The panel marked "Gel" is representative of the other lanes. The sizes of the AseI fragments (in kilobases) are shown on the left-hand side of the Gel strip. The gel bands are marked with arrows; white and black arrows indicate CI and CII bands, respectively. Bands without arrows are derived from other endogenous replicons. The gel was blotted, and the filter was cut into strips. The strips were then probed with radiolabeled PCR products generated from the genes named at the top of the strips. The autoradiograph strips have been realigned with the Gel strip. Probes to the genes, rpsA1, trpF, aroR, and trpB showed strong hybridization to the 360-kb CII AseI band. The trpA probe showed strong hybridization to the 910-kb CI AseI band. Probes to the genes, trpC, trpD, trpE, and trpG showed strong hybridization to the 1105-kb CI AseI fragment. These data are in agreement with the findings of Tn5 mapping.

It was noted that some of the probes hybridized less strongly, but visibly, to other AseI fragments. To determine if there were additional silent copies of these genes, we used them to probe regular (non-TAFE) BamHI and EcoRI genomic Southern blots (not shown). The results firmly indicated that in the TAFE Southern blots we were observing nonspecific hybridization to abundant, large TAFE fragments. In standard Southern blots, we could detect only single copies of all the genes described.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Transposon mutagenesis was used to generate R. sphaeroides auxotrophs with a Trp^- phenotype. The sites of Tn5 insertion were determined by TAFE gel electrophoresis and were mapped to CI and CII. These results suggested that the genes encoding the tryptophan biosynthetic pathway were distributed between the two chromosomes of this multichromosomal bacterium. Sequencing of the regions around the sites of Tn5 insertion indicated that transposons had disrupted the CI genes trpE, trpD, and ybaU, as well as the CII gene trpB. The insertions in ybaU are thought to have resulted in a Trp^- phenotype because of polar effects on the downstream gene trpE. Additional sequencing revealed the genes trpG and trpC on CI and trpF on CII. Additional cloning indicated that trpA was located on the CI physical map 1.23 Mb (149° counterclockwise) from the other CI trp genes. This accounted for all the structural genes of the classical tryptophan pathway.

To further verify function, the genes trpA, trpE, and trpG were disrupted with an Sm/Sp cartridge. In each case the disruption led to a Trp^- phenotype, confirming the role predicted from sequence analysis. The disruption of trpE for a second time (the first being with Tn5) acted as a control for Tn5 mutagenesis. This result suggested that the Trp^- phenotypes were the result of the Tn5 insertions and had not arisen because of a mutation at a second chromosomal location. This was further confirmed by complementation of the TrpA^- and TrpB^- strains with cosmids carrying the wild-type genes. A mutation at a second location was unlikely to have been complemented by these cosmids. In addition, the finding that disruption of these genes resulted in auxotrophy suggested that these genes are found in single copy within the genome. Southern hybridization confirmed this finding and corroborated the location of the mapped insertions. This has led us to conclude that the structural genes corresponding to the tryptophan biosynthetic pathway are indeed distributed between the two chromosomes of R. sphaeroides 2.4.1.

A number of the trp genes have overlapping stop and start codons. Such an organization has been noted previously in other organisms; e.g., in E. coli, trpB and trpA coding regions on the polycistronic trp mRNA are separated by overlapping stop and start codons. Efficient translation of the trpA coding region is subject to translational coupling; i.e., maximal trpA expression is dependent on prior translation of the trpB coding region. Therefore, it is both possible and understandable that the gene pairs trpG-trpD and trpF-aroR are translationally coupled. However, it is less obvious why translational coupling (if it does indeed occur) would be present between trpC-moaC-moeA. These last two genes have been implicated in molybdopterin biosynthesis. We have found a second copy of moeA on CII, downstream of torA, a gene that encodes the molybdenum-containing enzyme TMAO reductase (N. MOUNCEY and S. KAPLAN, unpublished results).

In addition to trpF, aroR, and trpB, other genes, i.e., cmkA, rpsA1, and hip, were also mapped to CII. Southern hybridization suggested that rpsA1 is located only on this chromosome. Their low BLASTP scores, combined with the finding that they are in a similar genomic order as in other bacteria, suggests that these are bona fide genes. This further reinforces previous work that suggested that CII encodes functions typical of any other bacterial chromosome.

However, bacteria that possess multiple chromosomes are an enigma. What selects for the maintenance of a divided genome once such an event has occurred? The possession of two chromosomes surely increases the complexity of cell division, imparting an increased risk for genetic lesions and decreased fitness of daughter cells. This is particularly noticeable in R. sphaeroides, where partial loss of CII could lead to auxotrophy, or inability to carry out translation or genome replication.

Multiple chromosomes would also be expected to lead to an increase in the complexity of coordinated gene expression, as supported by current reasoning. For example, if we examine the enzyme tryptophan synthase, we see that in most bacteria it is encoded by a trpBA operon. This "makes sense" because the cell needs to make equimolar amounts of the - and ß-subunits to form the functional heterotetrameric (₂ß₂) enzyme. In these bacteria, transcription of both genes is coordinated, and translation products are synthesized near each other to form the complete enzyme. This would appear to be an efficient and highly evolved process. However, in R. sphaeroides, the genes that encode these subunits are on different chromosomes. How does the cell ensure that the gene products are formed in equimolar amounts and in relative proximity for subunit association? Perhaps it does not, at least not to the same degree as in other bacteria. It may be that making different amounts of the two products does not decrease the fitness of the cell sufficiently for there to have been a strong selective drive toward operon formation. Therefore, in R. sphaeroides, the trp genes may be organized in a more ancient and perhaps less stringently regulated topology than normally seen in bacteria. Reinforcing this hypothesis is the finding that in Aquifex aeolicus, a member of the deepest branching family within the bacterial domain, the trp genes are distributed as individual genes throughout the genome (DECKERT et al. 1998 ). Indeed, A. aeolicus is extreme in that no two amino acid biosynthetic genes are found in the same operon. If the "disbursed genes are ancient and operons are recent" argument is accepted, then by analogy, having multiple chromosomes may also represent a more ancient and less "streamlined" genomic organization. Multiple chromosomes may persist, not because such an organization confers a biological advantage, but rather because it does not confer a sufficient decrease in fitness to have been selected against and lost from the genome pool.

Other members of the -Proteobacteria, e.g., Agrobacteria and Brucella, also have genomes comprising multiple chromosomes (or, as in the case of Rhizobia, megaplasmids), and it has been noted that many of these have infectious associations with eukaryotes. Evidence from 16S ribosomal RNA suggests that ancient members of this group gave rise to eukaryotic plastids, such as the chloroplast and mitochondrion (GRAY 1993 ). Therefore, complex genomic organization with "disbursed" as opposed to "operonic" or "condensed" gene organization may have been a prerequisite for the formation of early eukaryotic cells.

In R. sphaeroides, it is known that extensive gene duplication occurs between the two chromosomes. If a Rhodobacter-like organism was the progenitor of such plastids, then gene duplication could have provided the opportunity for the development of complex regulatory mechanisms, such as those found in eukaryotes. Such duplications may have permitted the evolution of differential regulation of each copy of the duplicated gene, resulting in a wider spectrum of conditions under which a gene or group of genes with similar function could be expressed.

The possession of multiple chromosomes also may have assisted in genetic export and exchange between the early plastids and their host, perhaps explaining why many plastid genes are encoded in the nucleus of modern eukaryotes. This suggests that if we examined the plastids of primitive unicellular eukaryotes, we may see remnants of their ancient bacterial origins. It is therefore intriguing that in the "primitive" unicellular red alga Cyanidium caldarium, the trpA gene is found on the plastid genome. In contrast, trpB is located in the cell nucleus (OHTA et al. 1994 ). Thus, the very unique structure of the R. sphaeroides genome, the dispersal of essential genetic loci between linkage groups, and the organism's unique position within the -Proteobacteria suggest its centrality to the origins of primitive eubacteria and plastids.

	ACKNOWLEDGMENTS

We are grateful to Agnes Puskas, Renata Ng, and David Needleman for their help with DNA sequencing; Mark Gomelsky for technical tips; and Jesus Eraso for technical tips and correcting our manuscript. We are also grateful to Robert Hazelkorn for providing us with the R. capsulatus trpA subclones and sequence before their publication. We also thank our reviewers. Their constructive criticism led to a greatly improved manuscript. This work was supported by National Institutes of Health grant GM-55481.

Manuscript received March 12, 1999; Accepted for publication June 9, 1999.

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