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Journal of Bacteriology, January 2004, p. 235-239, Vol. 186, No. 1
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.1.235-239.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Null Mutation of HvrA Compensates for Loss of an Essential relA/spoT-Like Gene in Rhodobacter capsulatus

Shinji Masuda1* and Carl E. Bauer2

Laboratory for Photobiology, RIKEN Photodynamics Research Center, Sendai 980-0845, Japan,1 Department of Biology, Indiana University, Bloomington, Indiana 474052

Received 4 August 2003/ Accepted 3 October 2003


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ABSTRACT
 
We report that a single relA/spoT-like gene exists on the Rhodobacter capsulatus chromosome, and its mutational loss is lethal. This gene could be mutated only under a mutational background of a null mutation in the nucleoid protein HvrA. This result suggests that there may be a direct link between HvrA-regulated promoters and the ppGpp-related stringent response.


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TEXT
 
The stringent response is a global regulatory system that controls gene expression in bacterial cells that are undergoing starvation for amino acids and/or carbon sources (for review, see reference 6). The stringent response has been extensively studied for almost 40 years, mainly in the {gamma}-proteobacterium Escherichia coli. These studies have established that the stringent response is mediated through the synthesis of guanosine 3',5'-bisdiphosphate (ppGpp) that is maintained in E. coli cells by two enzymes, RelA and SpoT. Amino acid limitation and the subsequent increase in the proportion of uncharged tRNA induce activation of a ribosome-bound ppGpp synthase called RelA. Biochemical and genetic analyses of the regulatory function of ppGpp have indicated that it controls the transcription of a large number of genes (e.g., rRNA operons and amino acid biosynthesis genes) through direct interaction of ppGpp with RNA polymerase (7, 24). Thus, changes in the amount of ppGpp rapidly send a signal to the transcriptional apparatus that there is a change in translational conditions. This allows cells to adapt to an environment of variable nutrient availability. The role of SpoT is to degrade ppGpp, which prevents prolongation of the stringent response. Under certain conditions, such as carbon deficiency, SpoT also exhibits ppGpp synthase activity, which is essential for maintaining the basal level of ppGpp that is needed for amino acid biosynthesis in E. coli (6).

Phylogenetic and biochemical studies indicate that there are three classes of bacterial RelA/SpoT orthologs: (i) ppGpp synthetase I, termed "RelA," which synthesizes ppGpp only during amino acid limitation; (ii) ppGpp synthetase II, termed "SpoT," which synthesizes ppGpp primarily during carbon limitation; and (iii) ppGpp synthetase III, termed "Rel," which synthesizes ppGpp after either amino acid or carbon limitation. SpoT and Rel, but not RelA, are also capable of hydrolyzing ppGpp in a manganese-dependent reaction (16, 27). From phylogenetic studies, a theory for the evolution of paralogous relA and spoT genes in proteobacteria was proposed, which suggests that there was an ancestral rel-like gene that underwent gene duplication, giving rise to the relA and spoT lineages that subsequently evolved into their specialized functions for ppGpp synthetase and hydrolase activity, respectively (16).

Recent genomic sequence data indicate that RelA/SpoT-like proteins are widely conserved in bacteria, with homologs also present in the higher plant Arabidopsis and in the green alga Chlamydomonas (13, 25). The Chlamydomonas RelA/SpoT-like protein exhibits ppGpp synthetase and hydrolase activities and was shown to localize in the chloroplast. The protoplast location suggests that the plant stringent response may be derived from a photosynthetic bacterial symbiont during evolution of the chloroplast. Genome sequencing indicates that RelA and SpoT homologs are exclusively found in the ß and {gamma} subdivisions of proteobacteria, whereas the Rel homologs are found in gram-positive bacteria, such as Bacillus, Clostridium, and/or Mycobacterium species. Phylogenetic analyses also suggests that the {alpha}-subdivision proteobacteria Bradyrhizobium japonicum, Caulobacter crescentus, and Rhodobacter capsulatus possess a unique relA/spoT-related gene that comprises a new branch on the RelA, SpoT, and Rel phylogenetic tree (16). Given that the subdivision of the {alpha} subgroup is earlier than that of ß- and {gamma}-subdivision event in the proteobacterial lineage (28), the RelA/SpoT-related proteins in the {alpha}-proteobacteria may retain characteristics that are ancestral to RelA and SpoT proteins. Thus, it was of interest to characterize the function of the relA/spoT-related genes in {alpha}-subdivision proteobacteria. In this study, we have chosen the {alpha}-proteobacterium R. capsulatus as a model organism to genetically characterize the unique relA/spoT-related branch. The advantage of R. capsulatus is that it exhibits remarkable bioenergetic versatility, allowing respiratory growth under aerobic (oxygen as an electron acceptor) and anaerobic conditions (dimethyl sulfoxide as an electron acceptor), as well as growth by photosynthesis (26). Genetic tools for this organism have also been well established (18). Our results indicate that R. capsulatus possesses only a single relA/spoT-related gene, which was found to be essential for its viability.

A single spoT-like gene is conserved in the {alpha} subdivision of proteobacteria. We identified relA/spoT homologous genes in several sequenced genomes of the {alpha}-subdivision proteobacteria by using a computer-aided similarity search. For this application, the amino acid sequence of the E. coli RelA protein was used as a homology probe of translated regions of sequenced genome from R. capsulatus, Mesorhizobium loti, B. japonicum, and Sinorhizobium meliloti. The sequence of R. capsulatus was obtained from the R. capsulatus genome project site at http://wit.mcs.anl.gov/WIT2/, and those of S. meliloti, M. loti, and B. japonicum were obtained from the Kazusa DNA Research Institute site at http://www.kazusa.or.jp/en/. In each organism, only a single open reading frame was found that exhibits similarity to the amino acid sequence of the E. coli RelA protein. Figure 1A shows a phylogenetic tree derived from deduced amino acid sequences of relA/spoT-related genes from various species. The tree was drawn with the programs ClustalX (22) and MEGA2 (15). All gaps in the sequence alignment were omitted in a pairwise manner, and the construction of the tree was performed by neighbor-joining methods (17). As suggested previously, putative RelA/SpoT-related proteins from {alpha}-proteobacteria form an independent cluster with closer similarity to SpoT than to RelA (16). An amino acid alignment of RelA, Rel, and SpoT homologs indicates that Rel and SpoT (but not RelA) conserve a part of the HD domain found in a superfamily of metal-dependent phosphohydrolases (1) that is thought to be involved in the ppGpp degradation (11, 13). The SpoT-like homologs in {alpha}-proteobacteria also have the HD domain (Fig. 1B). These observations suggest that {alpha}-proteobacteria contain a single SpoT-like protein that may be responsible for both synthesizing and hydrolyzing ppGpp, as is the case for SpoT and Rel proteins.



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  		FIG. 1. (A) Phylogenetic tree based on the amino acid sequences of RelA/SpoT/Rel homologs. The bootstrap values obtained by 1,000 replications are given beside the nodes. The accession numbers of the sequences used for sequence comparison and construction of the phylogenetic tree are as follows: Pseudomonas aeruginosa, AAG04323 and AAG08723; E. coli, J04039 and P17580; Xylella fastidiosa, AE003964 and AE003887; Neisseria meningitides, CAB85211 and CAB85138; Streptococcus pyogenes, AAK34667; Streptococcus pneumoniae, AAL00291; Enterococcus faecalis, AAO81720; Bacillus subtilis, U86377; C. crescentus, AAK23532; and Arabidopsis thaliana, AAD25787. The cluster of single spoT-like gene conserved in {alpha}-proteobacteria is indicated as SpoT*. (B) Partial amino acid sequence alignment of SpoT-like protein from R. capsulatus (R.cap), S. meliloti (S.mel), B. japonicum (B.jap), and M. loti; RelA and SpoT proteins from E. coli; and Rel protein from B. subtilis. The HD domain thought to be involved in the ppGpp degradation activity is boxed (see the text). The conserved amino acids are indicated by asterisks.

Previously, Mittenhuber (16) suggested that an ancestral spoT gene was duplicated in the ß and {gamma} branches of the proteobacteria and that the duplicated copy subsequently lost ppGpp-hydrolyzing activity and then adapted ppGpp synthesis to amino acid starvation (16). It was proposed that the duplicated copy then evolved to form the relA genes found in ß- and {gamma}-proteobacteria (16). However, our phylogenetic tree in Fig. 1 indicates that spoT/relA must have diverged before the separation of gram-positive bacteria and proteobacteria. This indicates that the ancestor to {alpha}-proteobacteria most likely had a relA gene that was lost after separation from the ß and {gamma} branches.

Disruption of the R. capsulatus spoT gene is lethal. To investigate the biological function of the spoT gene in R. capsulatus, we first constructed a suicide plasmid that contained a tetracycline resistance (Tcr) cassette (9) that replaces amino acids 140 to 623 of the R. capsulatus spoT coding region. The suicide vector used for mutagenesis was constructed as follows. First, two 600-bp DNA fragments corresponding to N- and C-terminal regions of the R. capsulatus spoT gene were amplified by PCR using the following two sets of primer pairs, respectively: RelA-F1 (5'-GGGAGCTCATGATCGATGTCGAAGACCTG-3') and RelA-R1 (5'-GGGATATCCCGGGCAAGCTTGACC-3') and RelA-F2 (5'-GGGATATCATCGGGCTTGCCGCGGATC-3') and RelA-R2 (5'-GGTCTAGAAGCAGCCGGTGCAGATGTTC-3'). RelA-F1 and RelA-R2 have a SacI and XbaI restriction site, respectively, whereas, both RelA-R1 and RelA-F2 have EcoRV restriction sites (underlined). These two fragments were digested with SacI, EcoRV, and XbaI and then cloned into a gentamicin resistance (Gmr) suicide vector, pZJD29A (Z. Jiang and C. E. Bauer, unpublished strain construction) at its SacI and XbaI sites with the SmaI-digested Tcr cassette (9). The resulting Tcr Gmr suicide vector was transferred to R. capsulatus wild-type strain SB1003 by using S17-1, an E. coli mobilizing strain (19) with single-crossover recombinants obtained at a frequency of 2 x 10-7 per recipient (single crossovers were selected for tetracycline [1 µg/ml] and gentamicin [10 µg/ml] resistance). The single-crossover recombinants were then grown for several generations with no gentamicin and subsequently plated on medium containing tetracycline and 5% sucrose. Because the suicide vector also encodes a sacB gene coding for levansucrase, which causes cell lethality when grown in the presence of sucrose (10), recombinants that undergo a second chromosomal cross that leads to loss of the plasmid with retention of chromosomal spoT::Tcr can be directly selected by growth on tetracycline and sucrose followed by scoring for gentamicin sensitivity (Gms). Interestingly, when the single-crossover recombinants were grown without gentamicin and then plated onto plates containing tetracycline plus sucrose, we were unable to observe any recombinants that underwent a second genetic exchange. The failure to perform allelic replacement occurred regardless of growth medium (minimal versus complex medium) or physiological growth conditions (dark aerobic, dark anaerobic, or photosynthetic). These results indicated that the loss of function of the spoT-like gene in R. capsulatus is lethal.

The R. capsulatus spoT gene can be disrupted in an hvrA-null mutation background. It was recently shown that E. coli strains lacking the two nucleoid proteins H-NS and StpA have a slow-growth phenotype that can be suppressed by null mutations in spoT and relA genes (12). This result let us address whether the R. capsulatus spoT gene could be mutated in an R. capsulatus strain that is disrupted for a nucleoid-like protein. Genome sequence analysis indicates that this species codes for a single H-NS-like protein called HvrA (3, 5; data not shown). We attempted the same insertional mutagenesis of the spoT gene in an hvrA mutant strain, MS03 (5), using an identical suicide vector to test whether the hvrA mutation suppresses the lethality of the loss of function of spoT. In contrast to our failure to obtain double recombinants in wild-type cells, double-crossover events could be readily obtained when grow on medium containing tetracycline and 5% sucrose. Double recombination leading to proper integration of the Tcr cassette into the chromosomal copy of spoT in the hvrA-disrupted strain was confirmed by PCR analysis; the resulting recombinant was named "SM05." From these results, it was concluded that the R. capsulatus spoT gene is essential, but can be eliminated by a compensating null mutation in the hvrA gene.

In vivo ppGpp synthesis. We next determined the effect of a nonfunctional spoT gene on the ppGpp metabolism in the hvrA spoT double-mutant strain SM05 after subjecting the cells to stringent growth conditions. In this experiment, cells were grown in MOPS (morpholinepropanesulfonic acid) medium (4) containing 0.2% Casamino Acids, minerals, and vitamins (26) at 34°C under aerobic-dark respiratory conditions to an optical density at 660 nm (OD660) of 0.05. The cells were then labeled with H332PO4 (100 µCi/ml; Amersham Pharmacia Biotech) with further incubation to OD660 of 0.4. In vivo (p)ppGpp synthesis was induced by supplying 1 mM serine hydroxamate (SerOHX) (Sigma), which inhibits tRNASer aminoacylation (23). After further incubation for 1 h, the 32P-labeled cells were mixed with an equal volume of 8 M formic acid and subjected to extraction by three cycles of freezing and thawing. The extracts were centrifuged at 8,000 x g for 10 min, and the resulting supernatant was analyzed by thin-layer chromatography on PEI-cellulose (Merck) with 1.5 M KH2PO4. As shown in Fig. 2, before supplying SerOHX, there was no detectable (p)ppGpp found in the wild-type parent cell line SB1003, in the hvrA mutant strain MS03, and in the hvrA spoT double-mutant strain SM05 (lanes 1 to 3, respectively). However, after SerOHX induction of the stringent response, significant quantities of (p)ppGpp were observed in nucleotide extracts from strains SB1003 and MS03 (lanes 4 and 5, respectively). In contrast, there was no detectable (p)ppGpp accumulation in SM05 after addition of SerOHX (lane 6). We also found that shorter and/or longer incubation after addition of SerOHX (for 15 min and 2 h, respectively) results in no ppGpp accumulation in strain SM05 (data not shown). These findings indicate that synthesis of (p)ppGpp in R. capsulatus upon induction of a stringent response requires a functional spoT gene.



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  		FIG. 2. Autoradiography of PEI thin-layer chromatography of formic acid cell extract from R. capsulatus strain SB1003 (wild type), MS03 ({Delta}hvrA), and SM05 ({Delta}hvrA {Delta}spoT). Labeled mononucleotides from SerOHX-untreated (lanes 1 to 3) and SerOHX-treated (lanes 4 to 6) cultures are shown.

Involvement of R. capsulatus spoT gene in photosystem synthesis. The H-NS-like nucleoid protein, HvrA, was originally found to function as a trans-acting factor needed for optimal photosynthesis gene expression (5). Since our mutational analysis can compensate for a defect in ppGpp synthesis, there may be a functional overlap between genes that are controlled by HvrA and those controlled by ppGpp. Interestingly, strain SM05 ({Delta}hvrA {Delta}spoT) forms colonies that are completely devoid of pigmentation when cells are grown on rich medium (PY plates) (30) under aerobic-dark respiratory conditions (Fig. 3A). Even more surprisingly, the pigmentation-defective phenotype of SM05 can be suppressed when these cells are grown under anaerobic (photosynthetic) conditions (Fig. 3B). Additionally, supplementation with a carbon source (e.g., glucose, malate and/or succinate) results in the comparable photopigment synthesis under aerobic conditions (Fig. 3C). Given that SM05 has the ppGpp0 phenotype (Fig. 2), these results indicate that basal levels of ppGpp produced by SpoT protein are required for aerobic photosystem synthesis in the absence of a rich carbon source in R. capsulatus.



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  		FIG. 3. Growth and colony pigmentation of R. capsulatus strain SB1003 (wild type), MS03 ({Delta}hvrA), and SM05 ({Delta}hvrA {Delta}spoT). (A) Growth on a PY medium (30) without glucose under aerobic-dark respiratory conditions. (B) Growth on a PY medium without glucose under anaerobic-light photosynthetic conditions. (C) Growth on a PY medium with 0.5% glucose under aerobic-dark respiratory conditions.

Concluding remarks. In this study, we found that mutational loss of a single spoT-like gene exhibits lethality in the {alpha}-proteobacterium R. capsulatus. In E. coli, spoT has also been shown to be essential for the growth. In this case, loss of SpoT results in prohibitively high intracellular levels of ppGpp that is constitutively produced by RelA, leading to cell death (21, 29). However, this is clearly not the case for the spoT mutation in R. capsulatus, which causes the inability to synthesize ppGpp (Fig. 2). Furthermore, there is no homologous relA gene on the sequenced genome of R. capsulatus. This suggests that there is a functional difference in the RelA/SpoT homologs in these two organisms.

The R. capsulatus spoT-like gene was successfully disrupted in the hvrA mutant strain, suggesting a link between ppGpp-related regulatory pathway and the function of the nucleoid protein HvrA. The hvrA-spoT double-mutant strain has an unusual phenotype of being unable to synthesize photopigments under dark semiaerobic conditions (Fig. 3A). The mechanism for the conditional phenotype on photosystem synthesis in SM05 is open to speculation. In E. coli, a direct link between ppGpp regulons and the function of nucleoid proteins H-NS and StpA was reported (12). It was shown that the level of ppGpp normally present in the cells has a regulatory role for certain promoters, and the effect of the molecule is influenced by the supercoiled state of these promoters that is controlled by the function of H-NS and StpA (12). It therefore seems that ppGpp may have a regulatory role in the promoter activity that is coregulated by the nucleoid protein HvrA in R. capsulatus. Recent studies have shown that the role of HvrA in R. capsulatus is not limited to regulate photosynthesis gene expression, but HvrA is also involved in regulating nitrogen fixation, ubiquinol oxidase and cytochrome oxidase genes (14, 20). In addition, expression of HvrA is regulated by the global redox-responsible two-component system, RegA and RegB (2, 8). Perhaps HvrA and SpoT-like proteins are functionally linked in this bacterium to efficiently capture the available energy source under different environmental changes (e.g., oxygen, light, nitrogen, amino acids, and/or the carbon source). Clearly, further biochemical and genetic analyses of the R. capsulatus spoT-like gene are needed to test this hypothesis, and such studies are currently under way.


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ACKNOWLEDGMENTS
 
S. M. thanks T. Koshiba, Tokyo Metropolitan University, for thin-layer chromatography equipment.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory for Photobiology, RIKEN Photodynamics Research Center, Sendai 980-0845, Japan. Phone: (81) 22-228-2047. Fax: (81) 22-228-2045. E-mail: smasuda{at}postman.riken.go.jp. Back


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Journal of Bacteriology, January 2004, p. 235-239, Vol. 186, No. 1
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.1.235-239.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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