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Journal of Bacteriology, December 2007, p. 8474-8483, Vol. 189, No. 23
0021-9193/07/$08.00+0     doi:10.1128/JB.00894-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Myxococcus xanthus Nla4 Protein Is Important for Expression of Stringent Response-Associated Genes, ppGpp Accumulation, and Fruiting Body Development ^,

Faisury Ossa,^1, Michelle E. Diodati,^2,, Nora B. Caberoy,¹ Krista M. Giglio,¹ Mick Edmonds,^1,¶ Mitchell Singer,^2* and Anthony G. Garza^1*

Department of Biology, Syracuse University, Syracuse, New York 13244,¹ Section of Microbiology, University of California, Davis, Davis, California 95616²

Received 7 June 2007/ Accepted 4 September 2007

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Changes in gene expression are important for the landmark morphological events that occur during Myxococcus xanthus fruiting body development. Enhancer binding proteins (EBPs), which are transcriptional activators, play prominent roles in the coordinated expression of developmental genes. A mutation in the EBP gene nla4 affects the timing of fruiting body formation, the morphology of mature fruiting bodies, and the efficiency of sporulation. In this study, we showed that the nla4 mutant accumulates relatively low levels of the stringent nucleotide ppGpp. We also found that the nla4 mutant is defective for early developmental events and for vegetative growth, phenotypes that are consistent with a deficiency in ppGpp accumulation. Further studies revealed that nla4 cells produce relatively low levels of GTP, a precursor of RelA-dependent synthesis of (p)ppGpp. In addition, the normal expression patterns of all stringent response-associated genes tested, including the M. xanthus ppGpp synthetase gene relA, are altered in nla4 mutant cells. These findings indicate that Nla4 is part of regulatory pathway that is important for mounting a stringent response and for initiating fruiting body development.

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In soil, vegetative swarms of Myxococcus xanthus feed on prey bacteria to obtain amino acids, which are used as a source of carbon, nitrogen, and energy (2, 10). When deprived of amino acids, large groups of M. xanthus cells migrate to aggregation centers and begin building multicellular fruiting bodies. Once a fruiting body is formed, rod-shaped cells within this structure differentiate into spherical, stress-resistant spores (for reviews, see references 11 and 62).

Amino acid starvation in M. xanthus cells leads to accumulation of the intracellular starvation signal (p)ppGpp and induction of an adaptive response known as the stringent response (52, 53, 63). In Escherichia coli, the ribosome-associated RelA protein catalyzes production of (p)ppGpp molecules that are capable of altering the promoter specificity of RNA polymerase (for reviews, see references 4 and 5). A mutation in the M. xanthus relA gene blocks (p)ppGpp synthesis, and cells carrying this mutation fail to initiate early developmental events (24). Furthermore, Singer and Kaiser (63) showed that ectopic expression of the E. coli relA gene causes M. xanthus cells to initiate early developmental gene expression in the absence of starvation. These findings indicate that (p)ppGpp accumulation is necessary and sufficient to initiate the M. xanthus developmental cycle.

Recent studies suggest that SocE inhibits (p)ppGpp production when nutrients are plentiful and that CsgA is required for maintaining relatively high levels of (p)ppGpp under starvation conditions (6, 7). Thus, it appears that there are both positive and negative input signals that modulate RelA-mediated synthesis of (p)ppGpp in M. xanthus.

After (p)ppGpp levels rise and the developmental process begins, M. xanthus cells produce a series of cell-cell developmental signals (9, 22, 40, 45, 49). Of these cell-cell developmental signals, the two that have been studied the most extensively are A-signal and C-signal. A-signal is produced early in development, and it serves as an indicator of cell density (46, 47), allowing M. xanthus to determine whether a sufficient number of cells are present to build a multicellular fruiting body. In contrast, C-signal is a contact-stimulated cell-cell signal that guides the aggregation and sporulation stages of M. xanthus development (30, 36-38, 50, 51). It is believed that M. xanthus uses these (p)ppGpp-dependent cell-cell signals to coordinate the large-scale changes in gene expression that occur during fruiting body development.

Many early developmental genes have ⁵⁴-like promoters (14, 15, 19, 20, 34, 41, 59). Transcription at ⁵⁴ promoters requires ⁵⁴-RNA polymerase and an enhancer binding protein (EBP). EBPs are often components in signal transduction pathways, and upon activation, EBPs help ⁵⁴-RNA polymerase form a transcription-competent open promoter complex (for reviews, see references 54, 65, and 73). In the last 10 years, 17 EBPs that are required for fruiting body development to proceed normally have been uncovered (3, 18, 21, 23, 28, 29, 32, 39, 66, 71), indicating that the ⁵⁴ system plays an important role in expression of M. xanthus developmental genes. In addition to its role in activating developmental genes, the ⁵⁴ system regulates expression of M. xanthus genes that are important for growth (8, 18, 35).

Recently, Caberoy et al. (3) showed that a mutation in the EBP gene nla4 affects fruiting body formation and sporulation. Here we show that an nla4 mutant accumulates relatively low levels of the stringent nucleotide ppGpp. We also show that the nla4 mutant is defective for early developmental events and for vegetative growth, phenotypes that are consistent with a deficiency in ppGpp accumulation. Further studies revealed that nla4 cells produce relatively low levels of GTP, a (p)ppGpp precursor. In addition, we found that expression of relA and of other stringent response-associated genes is altered in nla4 cells. These results indicate that Nla4 is part of the regulatory pathway that is important for mounting a stringent response.

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Bacterial strains and plasmids. The strains and plasmids used in this study are shown in Table 1. M. xanthus strain DK1622 (31) is wild type for growth, fruiting body development, sporulation, and motility. AG304 is a derivative of DK1622 (3) that carries the pNBC4 plasmid (which confers resistance to kanamycin) insertion in the EBP gene, nla4. To examine developmental gene expression in the presence of an nla4 mutation, plasmid pNBC29 (which confers resistance to oxytetracycline) was introduced into lacZ transcriptional reporter strains DK4300 (sdeK::4408 Tn5lacZ) and DK4521 (spi::4521 Tn5lacZ), creating strains AG375 and AG376, respectively. DK101, which carries the sglA1 (pilQ) mutation that allows dispersed growth in liquid media, forms fruiting bodies on agar surfaces but not in submerged cultures (27, 43, 69). Strain AG337 is a derivative of DK101 that carries the pNBC4 plasmid insertion in the nla4 gene.

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TABLE 1. Bacterial strains and plasmids used in this study

Media used for growth and development. M. xanthus strains were grown at 28 or at 32°C in CTT broth (1.0% Casitone, 10 mM Tris-HCl [pH 8.0], 1 mM KH₂PO₄, 8 mM MgSO₄), in CTTYE broth (CTT containing 0.5% yeast extract), or on plates containing CTTYE broth and 1.5% agar. CTT broth, CTTYE broth, and CTTYE agar plates were supplemented with 40 µg of kanamycin sulfate/ml or 10 µg of oxytetracycline/ml as needed. CTT soft agar contained CTT broth and 0.7% agar.

Fruiting body development was carried out at 32°C on plates containing TPM buffer (10 mM Tris-HCl [pH 8.0], 1 KH₂PO₄, 8 mM MgSO₄) and 1.5% agar. A-factor assays were performed in microtiter plates containing MC7 starvation buffer (10 mM morpholinepropanesulfonic acid [MOPS], 1 mM CaCl₂; final pH, pH 7.0).

E. coli strains were grown at 37°C in LB broth containing 1.0% tryptone, 0.5% yeast extract, and 0.5% NaCl or on plates containing LB broth and 1.5% agar. LB broth and LB agar plates were supplemented with 40 µg of kanamycin sulfate/ml or 10 µg of oxytetracycline/ml as needed.

M. xanthus development. M. xanthus strains were inoculated into flasks containing CTTYE broth, and the cultures were incubated at 28 or 32°C with vigorous swirling. After the cultures reached a density of 5 x 10⁸ cells/ml, the cells were pelleted, the supernatants were removed, and the cells were resuspended in TPM buffer to a density of 5 x 10⁹ cells/ml. Aliquots (20 µl) of the cell suspensions were spotted onto TPM agar plates and incubated at 32°C. M. xanthus cells were harvested at various times during development on TPM agar and used for ß-galactosidase assays or Western blot analysis as described below.

For real-time quantitative PCR (QPCR), cells were grown as described above, the cells were pelleted, the supernatants were removed, and the cells were resuspended in MC7 buffer to a density of 2 x 10¹⁰ cells/ml. The suspensions of concentrated cells were placed in a petri dish containing 28 ml of MC7 buffer and incubated at 32°C. Cells were harvested at various times during development and processed as described below.

ß-Galactosidase assays. Cells were harvested at different times during development on TPM agar plates. The cells were resuspended in 400 µl of TPM buffer, quick-frozen in liquid nitrogen as described previously (14), and stored at –80°C. ß-Galactosidase assays were performed as described by Kaplan et al. (33). ß-Galactosidase specific activities were measured in wild-type and nla4 mutant cells containing developmentally regulated lacZ reporter gene fusions. ß-Galactosidase specific activity was expressed in nanomoles of o-nitrophenol produced per minute per milligram of protein.

A-factor assays. MC7 buffer conditioned by wild-type strain DK101 (nla4⁺ asgA⁺), AG337 (nla4 asgA⁺), or DK476 (nla4⁺ asgA) cells served as the source of A-factor. A-factor was isolated as described by Diodati et al. (8). DK4323, a test strain that is defective for A-factor production and that carries the A-factor-dependent spi::4521 Tn5lacZ reporter fusion, was prepared as described previously (24, 57). Test cell aliquots (25 µl; 1.25 x 10⁸ cells) were added to the wells of 24-well microtiter plates containing 400 µl of conditioned MC7 buffer. The microtiter plates were incubated at 32°C, and the cells were harvested at various time intervals and assayed for ß-galactosidase activity as described above. One unit of A-factor activity is defined as the amount required to stimulate test cells to produce 1 U of ß-galactosidase activity.

Western blots. Approximately 1 x 10⁹ M. xanthus cells were harvested from TPM agar and placed in a lysis buffer (Tris-sodium dodecyl sulfate [pH 7.2])-protease inhibitor cocktail (Sigma). The cell suspensions were vortexed for 30 s, an equal volume of loading buffer (60) was added to each suspension, and the cell mixtures were boiled for 10 min. Samples containing equal amounts of protein were separated by electrophoresis through a 12% polyacrylamide gel and transferred to an Immobilon P membrane (Millipore) using a semidry blotting apparatus (Bio-Rad). The blots were probed with anti-FruA antibody or anti-CsgA antibody, followed by incubation with peroxidase-conjugated goat anti-rabbit immunoglobulin G (Boehringer Mannheim). Blots were developed using the Renaissance chemiluminescence reagent (NEN Life Science Products) and Amersham autoradiography Hyperfilm-MP.

Analysis of nucleotide pools. ³²P-labeled nucleotides were isolated and separated by thin-layer chromatography as described previously (52, 63). Labeled nucleotides were visualized using a STORM phosphorimaging scanner, and the relative nucleotide levels were determined using Image Quant software (Molecular Dynamics). Strains DK1622 and AG304 yielded results similar to those obtained for DK101 and AG337, respectively.

Real-time QPCR. Total cellular RNA was isolated from 2 x 10¹⁰ quick-frozen cells using the hot phenol method (60) and used to generate cDNA as described by Lancero et al. (48). Briefly, cDNA was generated from 1.5 to 2.0 µg of an RNA sample using 1 µl of Superscript III reverse transcriptase (Invitrogen) and 250 ng of random hexamers (Amersham Biosciences). The subsequent 16-µl PCR mixtures contained 0.5-µl aliquots of the cDNA synthesis reaction mixtures, gene-specific forward and reverse primers (1 µM), and 7.5 µl of iQ SYBR green Supermix (Bio-Rad). Primers were designed to yield approximately 100-bp PCR products. The primers used for QPCR are listed in Table S1 in the supplemental material. QPCR was performed using the iCycler iQ system from Bio-Rad. The rate of PCR-generated DNA accumulation was measured by continuous monitoring of SYBR green I (Molecular Probes) fluorescence. To confirm that RNA samples were not contaminated with residual genomic DNA, control cDNA synthesis reactions without reverse transcriptase were performed, and the synthesis reaction mixtures were analyzed using QPCR as described above for the test samples. The relative levels of relA, socE, and MXAN_1364 (Mx_1594) expression in wild-type cells and nla4 mutant cells were obtained using the relative standard curve method (user bulletin no. 2, Applied Biosystems) or as described previously (48). Standard curves were generated using 16S rRNA primers and various concentrations of wild-type cDNA (10^–7 to 10 ng). The expression level of relA, socE, or MXAN_1364 in each test sample was normalized to that of 16S rRNA and compared to the expression level in vegetatively growing wild-type cells (zero time).

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Expression of MXAN_2515 (Mx_838) and MXAN_2514 (Mx_837) in nla4 mutant cells. Previously, we showed that an insertion in nla4 causes aggregation and sporulation defects (3). The DNA sequence of the nla4 locus (17) places nla4 in close proximity to the downstream MXAN_2515 gene (Fig. 1). MXAN_2515 and nla4 are separated by approximately 160 bp, suggesting that these two genes are not part of the same operon and that the insertion in nla4 is unlikely to have a polar effect on MXAN_2515 transcription. However, since an insertion in MXAN_2515 yielded developmental phenotypes similar to that of the nla4 insertion (3), we wanted to confirm that the nla4 insertion does not affect MXAN_2515 transcription. To do this, we monitored MXAN_2515 expression in nla4 cells and wild-type cells during vegetative growth and development using QPCR analysis. We found that the levels of MXAN_2515 expression in wild-type cells and nla4 mutant cells were similar (data not shown). Furthermore, we found that expression of MXAN_2514, the gene immediately downstream of MXAN_2515, was similar in wild-type cells and nla4 cells (data not shown). These data indicate that the developmental defects of the nla4 mutant are due to inactivation of the nla4 gene.

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FIG. 1. Schematic diagram of the nla4 locus. The arrows show the locations of the indicated genes and the predicted directions of gene transcription. The designations of genes in the nla4 locus and the potential functions of the proteins that they encode were obtained from reference 17. Approximately 160 bp separates nla4 and the downstream gene, MXAN_2515. The inverted triangle indicates the position of the pNBC4 and pNBC29 plasmid insertions in the nla4 gene.

Expression of early developmental genes. As described by Caberoy et al. (3), aggregation of the nla4 mutant is delayed and incomplete, and this strain shows a dramatic drop in sporulation efficiency relative to wild-type cells. This strong aggregation defect suggests that the developmental process in the nla4 mutant goes awry early. To determine whether the nla4 mutation affects early developmental gene expression, we used Tn5lacZ transcriptional fusions to sdeK and spi, genes that are activated shortly after development is induced by ppGpp accumulation (41, 63). Expression of sdeK::Tn5lacZ and spi::Tn5lacZ in wild-type and nla4 mutant cells was monitored using ß-galactosidase assays as described previously (33). The peak expression of sdeK::Tn5lacZ in nla4 mutant cells was about 42% of the peak expression in wild-type cells (Fig. 2A), whereas the peak expression of spi::Tn5lacZ in nla4 cells was about 26% of the peak expression in wild-type cells (Fig. 2B). These findings indicate that the nla4 mutation alters gene expression early in development. Furthermore, these data show that the nla4 mutation affects expression of the key early developmental gene sdeK; sdeK codes for a histidine kinase that is important for the aggregation and sporulation phases of development (14, 42, 58). Another early developmental gene that is known to be important for aggregation and sporulation is fruA. FruA is a response regulator that plays an important role in the C-signaling pathway (12, 55, 64). To determine whether the nla4 mutation affects the levels of FruA in developing cells, wild-type cells and nla4 mutant cells were harvested after 18 h (when FruA levels peak) and 24 h of development on TPM agar, the cells were lysed, and whole-cell extracts were probed with anti-FruA antibody. In contrast to wild-type cells, no FruA was detected in nla4 cells at either the 18- or 24-h developmental time point (Fig. 3).

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FIG. 2. Developmental expression of sdeK and spi in wild-type and nla4 mutant cells. Expression of sdeK and spi was monitored at various times during development on TPM agar using the sdeK::4408 Tn5lacZ and spi::4521 Tn5lacZ reporter gene fusions, respectively. Mean ß-galactosidase specific activities were determined from three replicates. The error bars indicate standard deviations of the means. (A) ß-Galactosidase specific activity for strain DK4300 (nla4+ sdeK::4408 Tn5lacZ) () and strain AG375 (nla4 sdeK::4408 Tn5lacZ) (). (B) ß-Galactosidase specific activities for strain DK4521 (nla4+ spi::4521 Tn5lacZ) () and strain AG376 (nla4 spi::4521 Tn5lacZ) ().

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FIG. 3. FruA protein levels in wild-type cells and nla4 mutant cells developing on TPM agar. Whole-cell lysates were prepared from strains DK1622 (fruA+ nla4+), DK11063 (fruA nla4+), and AG304 (fruA+ nla4) at the indicated times during development. Samples containing 30 µg of total protein were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with anti-FruA antibody. The same total amount of protein was loaded into each lane. The experiment was repeated three times, and the results of a representative experiment are shown.

A-factor production. Early developmental genes, such as spi and fruA, are activated in response to A-factor (or A-signal) production. Given that the nla4 mutant fails to express normal levels of spi and FruA, we examined whether nla4 cells are defective for production of A-factor. The A-factor levels in the nla4 mutant were compared to those in wild-type cells and the asgA mutant, a strain that produces almost no A-factor (44). The peak levels of A-factor produced by nla4 mutant cells were about 14% of the peak levels produced by wild-type cells. The peak values, determined by measuring A-factor activity at various times during 24 h of development in submerged cultures, were 48.0 ± 5.9 U/ml for DK101 (asgA⁺ nla4⁺), 6.6 ± 0.6 U/ml for AG337 (asgA⁺ nla4), and 4.0 ± 0.6 U/ml for DK476 (asgA nla4⁺) (means ± standard deviations from three independent experiments). However, it did not appear that A-factor production was abolished in the nla4 mutant since the peak levels in this strain were 1.7-fold more than those in the asgA mutant. We concluded that the nla4 mutant has a strong defect in A-factor production, which is consistent with the defects in A-signal-dependent gene expression (Fig. 2B and 3). These data support the idea that Nla4 is important for events that occur very early in development, prior to the onset of aggregation and sporulation.

ppGpp and GTP accumulation. Accumulation of the intracellular starvation signal (p)ppGpp triggers A-signal production and early developmental gene expression (24, 63). Given that the nla4 mutation affects these (p)ppGpp-dependent events, we hypothesized that the nla4 mutant may be defective for (p)ppGpp accumulation. Therefore, we examined ppGpp levels in the nla4 mutant and wild-type cells. For these assays, wild-type cells and nla4 cells were grown in nutrient broth and subjected to a nutrient downshift, and the relative amounts of ppGpp were measured as previously described (8, 52, 63). The results of these studies are shown in Fig. 4A. In wild-type cells, the level of ppGpp increased about sixfold after 15 min of starvation, which was followed by a decrease to a new steady-state level (after 60 min) that was about fourfold higher than the vegetative growth level (zero time). During vegetative growth, the level of ppGpp in nla4 mutant cells was about 54% of the wild-type level. The peak ppGpp level in nla4 mutant cells was detected at 60 min poststarvation, compared to 15 min poststarvation in wild-type cells. In addition, the peak poststarvation level of ppGpp in nla4 cells was about 38% of that found in wild-type cells, and it did not increase significantly when the time of starvation was extended (data not shown). These results show that the level of ppGpp in nla4 cells is relatively low during vegetative growth and starvation. The ppGpp accumulation defect in nla4 cells is likely to be a major contributing factor in the downstream defects in early developmental gene expression and A-signal production.

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FIG. 4. Relative levels of ppGpp and GTP in wild-type and nla4 mutant cells. Nucleotides were isolated from DK1622 (nla4+ relA+) and AG304 (nla4 relA+) cells at different times following starvation and analyzed as described in Materials and Methods. The signal intensities were normalized to that of wild-type cells at zero time (vegetative growth). (A) Relative levels of ppGpp. (B) Relative levels of GTP. The assays were repeated four times, and the data for a representative sample are shown. The levels of ppGpp and GTP were similar when the nla4 insertion was placed in a DK1622 or DK101 strain background (data not shown). Strain MS1000 served as the negative control for the nucleotide assays (data not shown).

Since GTP is an essential precursor for RelA-dependent synthesis of (p)ppGpp (4, 63), we tested whether the nla4 mutant produces wild-type levels of GTP. The relative levels of GTP in nla4 mutant and wild-type cells are shown in Fig. 4B. The level of GTP in vegetatively growing nla4 cells was about 67% of that in wild-type cells. Under starvation conditions, the peak level of GTP in nla4 cells was about 53% of the peak wild-type level. These results indicate that nla4 mutant cells are defective for production of GTP.

Expression of genes implicated in ppGpp accumulation. One way in which Nla4 might influence ppGpp levels is by directly or indirectly regulating expression of stringent response-associated genes, such as relA, which encodes the sole M. xanthus ppGpp synthetase (17, 24, 63). To determine whether the nla4 mutation affects expression of relA, we monitored relA mRNA levels in wild-type and nla4 cells during vegetative growth and development using QPCR. We found that the level of relA mRNA in nla4 cells was about 16% of the level in the wild type during vegetative growth and that the peak level in nla4 cells during development was about 12% of the wild-type peak level (Fig. 5A). Since RelA is essential for synthesis of (p)ppGpp (24, 63), these results suggest that a major reason for the ppGpp accumulation defect in nla4 cells is that relA expression is severely impaired.

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FIG. 5. Expression of relA, MXAN_1364, and socE in wild-type and nla4 mutant cells. QPCR was used to examine developmental expression of relA (A), MXAN_1364 (B), and socE (C) in wild-type strain DK1622 () and nla4 mutant strain AG304 (). The expression of the relA, MXAN_1364, and socE genes was normalized to that of 16S rRNA. The indicated relA, MXAN_1364, and socE expression levels are relative to the levels found in vegetatively growing wild-type cells (zero time). The values are means derived from three replicates. The error bars indicate standard deviations of the means.

CsgA is required for maintaining relatively high levels of (p)ppGpp during fruiting body development, and developmental expression of the csgA gene is known to be dependent on a wild-type copy of relA and, presumably, the induction of a normal stringent response (7). Given that relA expression is reduced about six- to eightfold in the nla4 mutant, we speculated that developmental expression of csgA might be impaired or abolished in the nla4 mutant. Therefore, we examined the levels of CsgA in wild-type and nla4 mutant cells developing on TPM starvation agar. Cells were harvested after 18 h (when peak CsgA levels are observed [36]) and 24 h of development and were lysed, and whole-cell extracts were probed with anti-CsgA antibody. As shown in Fig. 6, no CsgA was detected in nla4 cells at 18 or 24 h of development. Furthermore, no CsgA was detected when nla4 mutant cells were given additional time to develop (data not shown).

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FIG. 6. CsgA protein levels in wild-type cells and nla4 mutant cells developing on TPM agar. Whole-cell lysates were prepared from strains DK1622 (csgA+ nla4+), DK5208 (csgA nla4+), and AG304 (csgA+ nla4) at the indicated times during development. Samples containing 30 µg of total protein were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with anti-CsgA antibody. The same total amount of protein was loaded into each lane. The experiment was repeated three times, and the results of a representative experiment are shown.

There are two additional M. xanthus genes that have been implicated in the stringent response, socE and MXAN_1364 (Mx_1594). The socE gene codes for a negative regulator of (p)ppGpp accumulation (6, 7). Hence, socE expression is relatively high during vegetative growth and relatively low during development. The product of the MXAN_1364 gene has similarity to the N-terminal hydrolase domain of E. coli SpoT (8; K. A. O'Connor and D. R. Zusman, personal communication), a protein that modulates (p)ppGpp levels in response to certain starvation cues (16). Based on this finding, it has been proposed that MXAN_1364 codes for a protein involved in the stringent response (8). To determine whether the nla4 mutation affects expression of MXAN_1364 and/or socE, QPCR was used to examine MXAN_1364 and socE mRNA levels in nla4 and wild-type cells during vegetative growth and development (Fig. 5). The level of MXAN_1364 mRNA in nla4 cells during vegetative growth was about 38% of the wild-type level, and the peak level in nla4 cells during development was about 52% of the wild-type peak level (Fig. 5B). As reported previously (7), socE mRNA levels were relatively high when wild-type cells were growing vegetatively and they declined when wild-type cells initiated development (Fig. 5C). A similar trend in socE expression was observed in nla4 cells (Fig. 5C). However, the socE mRNA levels in vegetatively growing nla4 cells were about eightfold higher than those found in vegetatively growing wild-type cells. Thus, the nla4 mutation affects expression of all stringent response-related genes that we tested in this study. The implications of these findings are addressed in the Discussion.

Vegetative growth. Low vegetative growth rates have been reported for relA and nla18 mutants, both of which accumulate very low levels of ppGpp (8, 24). When grown in CTT broth at 32°C, which are standard laboratory conditions, nla4 cells had a generation time of 12 to 16 h, whereas wild-type cells had a generation time of about 5 h. The vegetative growth defect of nla4 mutant cells was less severe when the cells were grown at 28°C and the CTT broth was supplemented with yeast extract (CTTYE broth). Under these conditions, nla4 mutant cells had a generation time of about 11 h, while wild-type cells had a generation time of about 5 h (Fig. 7). Thus, like other M. xanthus mutants that fail to mount a normal stringent response, the nla4 mutant is defective for growth.

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FIG. 7. Growth of wild-type and nla4 mutant cells. Cells were grown at 28°C in CTTYE broth. Representative growth curves produced by wild-type cells and by nla4 mutant cells are shown. In this experiment, the doubling time of wild-type cells was 5.1 h and the doubling time of nla4 mutant cells was 10.3 h. Based on data from several growth experiments, the average doubling time of wild-type cells is 5.0 ± 0.2 h and the average doubling time of nla4 mutant cells is 10.7 ± 0.6 h.

The nla4 mutant (AG304) used in the vegetative growth studies was generated by pNBC4 plasmid insertion in the nla4 gene (Table 1). Plasmid pNBC4 imparted kanamycin resistance to the nla4 mutant, and we found that to maintain the nla4 mutant phenotype, this strain had to be grown in nutrient broth containing kanamycin; nla4 cells rapidly lost kanamycin resistance and their vegetative growth defect when they were placed in nutrient broth without kanamycin. To confirm that the slow-growth phenotype of the nla4 mutant was not due to the presence of kanamycin in CTTYE broth, AG306 cells containing the pNBC6 plasmid insertion that conferred kanamycin resistance were grown in CTTYE broth supplemented with kanamycin and were shown to have typical wild-type doubling times, about 5 h (data not shown). Therefore, the slow-growth phenotype of nla4 mutant cells resulted from inactivation of the nla4 gene rather than the presence of the antibiotic.

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Fifty-two genes encoding EBPs were identified in the M. xanthus genome sequence (17), and most of these transcriptional activators have been characterized by mutational analyses (3, 18, 21, 23, 28, 29, 32, 39, 66, 71). Among the characterized EBP mutants, nla4 and nla18 mutants are the only mutants that have severe defects in vegetative growth and fruiting body development (3, 8). This phenotype suggests that the Nla4 and Nla18 regulatory pathways play unique roles in the transition from vegetative growth to development.

In this report, we focused on the effects that nla4 inactivation has on M. xanthus fruiting body development. Previous morphology studies suggested that fruiting body development goes awry relatively early in nla4 cells (3), and here we found that the nla4 mutation affects expression of early developmental genes. Two of these early developmental genes, sdeK and fruA, encode proteins that are important for downstream morphological events and for downstream changes in gene expression (12, 14, 42, 55, 58). In addition, we found that production of A-signal, a cell density signal that is required for expression of the vast majority of developmental genes that have been tested (45-47, 57), is almost completely abolished in nla4 cells. Given that the nla4 mutation has a strong effect on important early developmental events, perhaps it is not too surprising that nla4 cells fail to fully express genes that are induced during aggregation and sporulation (data not shown) and to aggregate and sporulate normally.

Since the M. xanthus early developmental pathway is activated by the accumulation of the intracellular starvation signal (p)ppGpp, the defects in early developmental gene expression and A-signal production are likely to be linked to the strong negative effect that the nla4 insertion has on ppGpp accumulation. We propose that the relatively low levels of ppGpp in nla4 cells are primarily due to (i) the six- to eightfold reduction in expression of the (p)ppGpp synthetase gene, relA, and (ii) the relatively low levels of GTP, which, among other things, serves as a (p)ppGpp precursor (4). In addition to the stringent response, low GTP levels might affect other cellular processes that are important for fruiting body development.

It seems likely that low levels of ppGpp and GTP contribute significantly to the vegetative growth defect of the nla4 mutant. There are precedents for vegetative growth defects in M. xanthus mutants that fail to mount a normal stringent response; vegetative growth defects have been previously reported for the relA and nla18 mutants, both of which have ppGpp accumulation defects (8, 24). Furthermore, GTP is known to be important for a variety of growth-related functions (13, 56, 61). The finding that Nla4 is important for vegetative growth is consistent with previous data showing that rpoN, which codes for ⁵⁴, is an essential M. xanthus gene (35).

The nla4 mutant's defect in expression of stringent response-associated genes is not limited to relA; the nla4 mutation alters expression of csgA and socE, genes whose protein products are known to be positive and negative regulators of (p)ppGpp accumulation, respectively (6, 7). It has been proposed that M. xanthus balances the levels of the CsgA and SocE proteins in order to modulate the stringent response. In cells that are maintaining vegetative growth, ppGpp levels are low. Hence, expression of the negative regulatory gene socE is relatively high and expression of the positive regulatory gene csgA is relatively low. The reverse is true for csgA and socE expression during fruiting body development, when ppGpp levels are high.

In cells carrying an nla4 mutation, the trend in socE expression is similar to that found in wild-type cells; socE expression is high during vegetative growth and decreases during development. However, the level of socE expression in vegetatively growing nla4 cells is about eightfold higher than the level of socE expression in their wild-type counterparts, indicating that the presence of the Nla4 protein has a negative effect on socE expression in vegetative cells. Since EBPs function as transcriptional activators, it is highly likely that this negative effect on socE transcription is indirect. The fact that the nla4 mutation abolishes CsgA expression in developing cells suggests that Nla4 is a positive regulator of csgA transcription during fruiting body development. It seems likely that the absence of detectable CsgA in nla4 cells is due at least in part to the six- to eightfold reduction in relA expression; developmental expression of the csgA gene is known to be relA dependent (7, 24). The idea that Nla4 regulates expression of csgA indirectly is consistent with evidence suggesting that csgA uses a ⁷⁰-type promoter (50).

Expression of MXAN_1364, a gene that is thought to play a role in the stringent response (8; O'Connor and Zusman, personal communication), is reduced in nla4 cells. The product of MXAN_1364 was tagged as a (p)ppGpp hydrolase based on DNA sequence analysis. This suggests that the role of the MXAN_1364 protein in the stringent response might be (p)ppGpp turnover, an idea that has yet to be examined experimentally. Studies in E. coli have shown that (p)ppGpp accumulation depends on the opposing synthetic and hydrolytic activities of the RelA and SpoT enzymes (16, 25, 26, 67, 68, 70, 72). Presumably, M. xanthus also accommodates the need for certain levels of (p)ppGpp by adjusting these activities accordingly. Since RelA and MXAN_1364 are predicted to have opposing activities, perhaps the relatively low levels of MXAN_1364 expression in nla4 cells is an adjustment to the relatively low levels relA expression.

Is it possible that Nla4 directly regulates expression of M. xanthus stringent response genes? The csgA gene does not appear to use a ⁵⁴-type promoter (50), indicating that csgA expression is indirectly regulated by Nla4. Given that EBPs are positive regulators of gene transcription and that Nla4 has a negative effect on socE expression, we believe that Nla4 is a indirect regulator of socE transcription as well.

Using total RNA isolated from vegetative cells, the primer extension studies of Harris et al. (24) uncovered a putative ⁷⁰-like promoter for the relA gene. This ⁷⁰-like promoter is located within the coding sequence of an upstream gene (MXAN_3203) that appears to be transcribed in the opposite orientation with respect to relA. When we scanned the MXAN_3203 gene for additional promoters using the PromScan bioinformatics tool (http://molbiol-tools.ca/promscan/) (1), a putative ⁵⁴-like promoter was found upstream of the ⁷⁰-like promoter identified by Harris et al. (24). As shown in Fig. 8, this putative ⁵⁴ promoter has the highly conserved GG dinucleotide in the –24 region and the highly conserved GC dinucleotide in the –12 region. Furthermore, five of seven nucleotides in the –24 region and four of five nucleotides in the –12 region of this promoter are identical to nucleotides found in the ⁵⁴ consensus sequence. These findings suggest that relA might use two different promoters, a ⁷⁰-like promoter and a ⁵⁴-like promoter. These findings also imply that Nla4 might directly regulate relA transcription, an idea that warrants further investigation.

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FIG. 8. Alignment of putative 54 promoters found upstream of the relA and MXAN_1364 genes. Putative 54 promoters for relA and MXAN_1364 were identified using the PromScan bioinformatics tool (http://molbiol-tools.ca/promscan/) and the M. xanthus genome sequence (17). Nucleotides in the putative relA and MXAN_1364 promoters that match those found in the 54 promoter consensus sequence are in bold type and are underlined. Y = C or T; R = A or G.

The promoter of MXAN_1364 has not been analyzed experimentally. However, when PromScan was used to examine the region upstream of MXAN_1364, a putative ⁵⁴-type promoter was identified (Fig. 8). We found the highly conserved GG dinucleotide in the –24 region of the putative MXAN_1364 promoter and the highly conserved GC dinucleotide in the –12 region of this putative promoter. The putative MXAN_1364 promoter shows a great deal of similarity to the ⁵⁴ consensus sequence, with six of seven nucleotides identical in the –24 region and four of five nucleotides identical in the –12 region. These findings suggest that Nla4 might directly regulate transcription of MXAN_1364. Additional work is needed to test this proposal and to identify the components in the signal transduction network that modulates the activity of Nla4.

 
We thank Lotte Søgaard-Andersen for providing CsgA and FruA antibodies. We also thank Roy Welch, Jimmy Jakobsen, Barry Goldman, Dale Kaiser, Monsanto Company, and TIGR for providing access to the M. xanthus genome sequence prior to GenBank submission (accession number CP000113). We thank M. M. Matteson for her valuable help with grammar.

This work was supported by Public Health Service grants T32GM0737 and GM56765B from the National Institute of General Medical Sciences to M. E. Diodati, by Public Health Service grant GM54592 from the National Institute of General Medical Sciences to M. Singer, and by National Science Foundation grant MCB 0615806 to A. Garza.

 
* Corresponding author. Mailing address for A. Garza: Department of Biology, Syracuse University, BRL Room 200, 130 College Place, Syracuse, NY 13244-1220. Phone: (315) 443-4746. Fax: (315) 443-2012. E-mail: agarza{at}syr.edu. Mailing address for M. Singer: Section of Microbiology, One Shields Avenue, University of California, Davis, Davis CA 95616. Phone: (530) 752-9005. Fax: (530) 752-9014. E-mail: mhsinger{at}ucdavis.edu

Published ahead of print on 28 September 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

M.D. and F.O. contributed equally to this work.

Present address: Department of Biological Sciences, Stanford University, 371 Serra Mall, Stanford, CA 94306.

^¶ Present address: Integrative Biomedical Sciences Program, The University of Alabama at Birmingham, 1670 University Boulevard, Birmingham, AL 35243.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Bacteriology, December 2007, p. 8474-8483, Vol. 189, No. 23
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