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Keywords:

  • Stress response;
  • RNA polymerase;
  • Sigma factor;
  • SigB;
  • Gene disruption;
  • Brevibacterium

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

We have previously cloned a gene encoding a SigB, a principal-like sigma factor in Brevibacterium flavum, which was induced by several stress conditions. To clarify the in vivo function of this sigma factor, the sigB gene was disrupted by a homologous recombination, replacing the internal essential coding region in B. flavum chromosome by a kanamycin resistance marker gene. This mutation dramatically decreased vegetative growth rates of B. flavum. Studies of the effect of the sigB mutation on growth and viability of the cells under conditions of stress showed that the sigB mutant had increased susceptibility to acid, salt, alcohol, heat and cold stress. The plasmid-born wild-type sigB gene complemented the mutation. Based on the results, we propose that SigB has a role in vegetative growth and in response to various environmental stresses.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Corynebacteria are Gram-positive, non-sporulating soil microorganisms and the non-pathogenic species, such as Corynebacterium glutamicum, Brevibacterium lactofermentum and Brevibacterium flavum are widely used for the industrial production of amino acids[1]. Bacteria are naturally exposed to various stresses. The response to these stress conditions is mediated by sigma factors of RNA polymerase, which govern expression of genes encoding stress proteins essential for overcoming these unfavorable conditions. Based on the sequence similarities, two major families are known to occur in eubacteria: σ70 and σ54 families[2]. The σ70 family has been divided into three groups: group 1 comprises primary sigma factors essential for cell viability, group 2 includes nonessential alternative sigma factors that are highly similar to group 1, and group 3 sigma factors are nonessential alternative sigma factors that vary more significantly in sequence from the other two groups[2]. Recently, a distantly related group 3 subfamily of sigma factors, so-called ECF (extra cytoplasmic function) sigma factors, has been characterized. Common feature of this subfamily is regulation of extracytoplasmic functions[2]. In Gram-negative bacteria (as the most studied strain Escherichia coli), the group 2 sigma factor, σS, has been shown to be a major regulator of starvation and stress response[3]. In contrast, in Gram-positive bacteria, the general stress response is governed by the alternative group 3 sigma factor σB[4]. Much has been learned about the general stress response sigma factor σB in Bacillus subtilis, and alternative sigma factors homologous to σB have also been found in other Gram-positive bacteria[4].

Up to now, very little information is available about stress response in corynebacteria. In our previous work, we cloned two sigma factor genes, sigA and sigB, from B. flavum CCM 251, coding for homologs of principal sigma factors of RNA polymerase. While expression of sigA was constitutive during growth and stress conditions, sigB was significantly induced by several stress conditions. The results suggested that SigA represents the functional group 1 principal sigma factor, and principal-like group 2 sigma factor SigB might have a function in stress response[5]. Sequence analysis of the recently sequences genome of the closely related strain Corynebacterium diphtheriae (http://www.sanger.ac.uk/Projects/C_diphtheriae/) revealed presence of SigA and SigB orthologues and seven ECF sigma factors. In the present study, we investigated the role of SigB in B. flavum by phenotypic analysis of a mutant with a disrupted sigB gene under several stress conditions. The results indicated that SigB has a role in vegetative growth, and it is involved in the response to several environmental stresses.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Bacterial strains, plasmids and culture conditions

B. flavum CCM 251 wild-type[6] was used in this study. E. coli XL1-Blue™ (Stratagene) was used as a host, and plasmids pBluescript II SK+ (Stratagene) and LITMUS 28 (New England Biolabs) were used for E. coli cloning experiments. E. coli ET12567[7] was used for preparation of non-methylated plasmid DNA. Plasmids pSigB0 and pSigB3, comprising the B. flavum sigB gene, came from our previous work[5]. pSigB0 contains a 1.8-kb Bam HI fragment of chromosomal DNA from B. flavum CCM 251 in pBluescript II SK+, and pSigB3 contains a 1.7-kb Cla I fragment in pBluescript II SK+[5]. The plasmid pJUP06 was prepared by deletion of a 3-kb Pst I fragment, containing E. coli origin of replication, of pJUP05[8]. Growth of B. flavum was carried out in Luria–Bertani (LB) medium at 30°C. Transformation of B. flavum was done by electroporation as described in[9]. Growth and transformation of E. coli were carried out according to[10].

2.2DNA manipulations and Southern blot hybridization

DNA manipulations in E. coli were done as described in[10]. Chromosomal DNA from B. flavum strains was prepared as described in[11]. For Southern blot hybridization, 1 μg of the chromosomal DNA was digested with an appropriate restriction endonuclease, separated by electrophoresis in 0.8% (w/v) agarose gel in TBE, and transferred on Hybond N (Amersham) as described in[10]. The membrane was hybridized with random-primed digoxigenin (DIG)-labelled DNA probe (Boehringer, Mannheim, Germany) according to the manufacturer's instructions. A 750-bp Cla I–Xho I DNA fragment (Fig. 1A) was used as a probe for hybridization experiments. A signal was detected by DIG chemiluminescent detection kit using CSPD (Boehringer, Mannheim, Germany). The membrane was immediately exposed to an X-ray film.

image

Figure 1. A: Physical maps of chromosomal DNA comprising the wild-type B. flavum sigB gene, and sigB-disrupted allele of the B. flavum BD2. The 1.6-kb Xho I–Bam HI DNA fragment of pSigB1, the 1.7-kb Cla I fragment of pSigB3, and the 3.6-kb Sal I–Xba I fragment of pSigB4 with flanking restriction sites from polylinker regions of cloning vectors are shown by stippled boxes. The bent arrow denotes the positions of the sigB promoter. The hatched box represents the aph gene. The black bar below the maps represents the probe (a 750-bp ClaXho I fragment) used for Southern hybridization analysis. Relevant restriction sites are indicated. B: Southern hybridization analysis of chromosomal DNA from gene replacement experiments. 1 μg of DNA from the corresponding strain was digested with the restriction enzymes indicated, separated by electrophoresis in 0.8% (w/v) agarose gel and transferred on Hybond N (Amersham) as described in[10]. Hybridization followed the standard DIG protocol (Roche, Mannheim, Germany) using the DIG-labelled probe (A). Lambda DNA-Mlu I digest was used as the size standard.

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2.3Construction of the B. flavum sigB insertional mutant

A 1.6-kb Xho I–Bam HI DNA fragment of pSigB0 was cloned into LITMUS 28 digested with Bgl II and Xho I, to create plasmid pSigB1 (Fig. 1A). The 1.6-kb Spe I–Eco RI DNA fragment from pSigB1 was successively cloned into pAPHII1[12] digested with Spe I and Eco RI, to generate plasmid pSigB2. A 700-bp Hin dIII–Kpn I fragment from pSigB3 was inserted into pSigB2 digested with Hin dIII and Kpn I, creating pSigB4, which contains the sigB allele disrupted by the kanamycin resistance gene aph (Fig. 1A). A 3.6-kb Sal I–Xba I fragment of pSigB4 containing the sigB-disrupted allele was cloned into pIJ666[13] digested with Sal I and Xba I. The resulting non-replicative plasmid pSigB5 was used to transform B. flavum CCM 251. For selection, the solid LB medium with 25 μg ml−1 kanamycin was used. Kanamycin-resistant clones were further analyzed for kanamycin resistance and chloramphenicol sensitivity that might indicate a double crossover event. The correct integration of the chloramphenicol-sensitive clones was confirmed by Southern blot hybridization.

2.4Complementation of the sigB mutation

A 1.7-kb Cla I fragment carrying the sigB gene from pSigB3 (Fig. 1A) was cloned into pJUP06 digested with Cla I, to generate pJUPB1, which was used in complementation studies. Before used for transformation of B. flavum BD2, the plasmid was passaged through the non-methylating E. coli strain ET12567[14]. The selection was done on plates with 10 μg ml−1 chloramphenicol. The presence of pJUPB1 in the clones was ascertained by isolation of this plasmid from all the clones.

2.5Application of different stress conditions

In stress resistance experiments the following stress conditions were applied to growing bacterial cultures 14 h after inoculation: for acid shock, 1 M HCl was added to shift pH of the culture from 7 to 4.4; for salt stress, solid NaCl was added to final concentration of 5% (w/v); for ethanol stress, ethanol was added to final concentration of 8% (v/v); for oxidative stress, H2O2 was added to final concentration of 30 mM; for heat shock, the culture was transferred to 39°C; for cold shock, the culture was rapidly chilled in water bath and the cultivation proceeded at 10°C. After application of stress conditions, the bacterial cultures were grown for additional 26 h, and the growth of bacteria was monitored by spectrophotometric measurements of OD570. Experiments were repeated at least three times, the values represent means of at least three experiments.

Viability of the cell cultures at certain time points was determined by plating of appropriate dilutions of growing cultures on LB agar. Simultaneously with stressed cultures, the growth and viability of parallel unstressed cultures of B. flavum CCM 251, B. flavum BD2 and B. flavum BD2 (pJUPB1) were monitored in each experiment. Survival of each culture after stress application was expressed as the percentage viability of parallel stressed and unstressed cultures at the same time point. Experiments were repeated, the values represent means of at least three experiments. Stability of the plasmid pJUPB1 in B. flavum BD2 was confirmed by plating of appropriate dilutions on LB agar with 10 μg ml−1 chloramphenicol.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Disruption of the sigB gene

To investigate the role of SigB ortholog in B. flavum CCM 251, a mutant strain with the disrupted sigB gene was prepared by homologous recombination. The kanamycin resistance gene (aph) was inserted between Xho I and Hin dIII sites, replacing 246 bp of the internal region of sigB (deleting amino acids 101–161) encoding the whole region 2, which is essential for function of sigma factors (Fig. 1A). The resulting plasmid pSigB5 (Section 2) contained an additional marker, the chloramphenicol resistance gene, and lacked any sequence necessary for replication in B. flavum. The plasmid was introduced into B. flavum CCM 251 competent cells by electroporation. Since pSigB5 was unable to replicate in B. flavum, kanamycin-resistant transformants were expected to arise from homologous recombination between B. flavum insert in pSigB5 and the corresponding region in the chromosome. Two types of clones were expected: kanamycin-resistant and chloramphenicol-resistant that might arise from a single crossover; and kanamycin-resistant and chloramphenicol-sensitive, arising likely from a double crossover between both flanking regions on both sides of the aph gene, resulting in the replacement of the wild-type sigB gene by the sigB-disrupted allele. Five kanamycin-resistant clones were identified. They were further analyzed for chloramphenicol sensitivity. Two chloramphenicol-sensitive clones were identified, and correct integration was confirmed by Southern blot hybridization (Fig. 1B). Both clones had similar phenotype and were designated as B. flavum BD1 and B. flavum BD2. Both mutant strains were viable and stable. By microscopic analysis, they were morphologically indistinguishable from the wild-type strain, B. flavum CCM 251. One clone, B. flavum BD2, was chosen for further stress resistance studies.

3.2Phenotypic analysis of the B. flavum sigB-disrupted strain

The growth of the sigB-disrupted B. flavum BD2 was compared with the wild-type parental strain B. flavum CCM 251. Interestingly, the mutant strain grew more slowly, and the maximal optical density at the stationary phase reached only 50%, compared with the wild-type strain (Fig. 2). Since unstressed sigB mutant strain did not decrease viability in stationary growth phase, we suggest that SigB is not essential for survival in stationary phase.

image

Figure 2. Role of sigB in the response to different environmental stresses. B. flavum CCM 251 (circles) and sigB mutant B. flavum BD2 (squares) were grown for 14 h at 30°C, then subjected to various environmental stresses, and the growth was monitored for further 26 h as described in Section 2. A: Effect of 30 mM H2O2, B: 5% (w/v) NaCl, C: 8% (v/v) ethanol, D: acid stress (1 M HCl was added to shift pH of the culture to 4.4), E: heat shock (39°C), F: cold shock (10°C). Open and solid symbols represent unstressed and stressed cultures, respectively. The arrows indicate the time points when stress conditions were applied.

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To study the effect of stresses on growth rate, the brevibacterial cultures were grown for 14 h (exponential phase) and then exposed to the stress conditions. Growth was monitored for further 26 h and survival measured as described in Section 2. Growth patterns of sigB mutant and the parental strain B. flavum CCM 251 were investigated after exposure to H2O2. Interestingly, even concentrations up to 30 mM did not affect growth of either strain (Fig. 2A). Using a paper disc method[15], the sensitivity of B. flavum BD2 and B. flavum CCM 251 to higher concentrations of H2O2 (0.3, 0.6, and 1.5 M) was checked. The concentrations of 0.6 and 1.5 M inhibited the growth of both strains, but no significant differences between these two strains were observed (data not shown). Similarly, the oxidative stress (30 mM H2O2) seemed to have no significant effect on viability of either wild-type or mutant B. flavum BD2 cells (Table 1). To prove that H2O2 was active, we performed similar experiments with the Gram-positive Streptomyces coelicolor A3(2) strain. The concentration as low as 3 mM inhibited growth of the strain (data not shown). Monitoring of the growth of the wild-type and sigB-disrupted strains in the presence of 5% NaCl revealed a decrease of the growth rate of B. flavum BD2, whereas the growth of the parental strain was not influenced (Fig. 2B). However, the cells survival under salt stress (Table 1) indicated less effect of the sigB gene disruption on cell viability (about 1.5-fold). Although 4% ethanol has no inhibitory effect for either wild-type or sigB-disrupted strains (data not shown), 8% ethanol dramatically influenced growth of both strains (Fig. 2C). However, by comparing the cell survival in 8% ethanol stress conditions we found a considerable difference (about 7.8-fold) between the mutant and parental strains (Table 1). By shifting the pH of B. flavum CCM 251 and B. flavum BD2 cultures to 4.4, the growth of both cultures was markedly inhibited. However, in contrast to the sigB mutant, where shift to acid pH 4.4 has stopped the growth, the growth rate was only decreased for wild-type strain (Fig. 2D). Moreover, the pH value of the culture of B. flavum CCM 251 increased in the course of continued cultivation to 5.3, in contrast to the sigB mutant, where the pH of the culture remained 4.4 during the entire cultivation period. Acid stress also considerably influenced the viability of both mutant and parental strain, but survival of B. flavum BD2 was markedly lower than survival of B. flavum CCM 251 (Table 1). From growth patterns of the wild-type and sigB-disrupted strains after exposure to 39°C we can see that the heat shock influenced markedly growth of both cultures (Fig. 2E). However, the mutant strain stopped growth almost immediately, and the parental wild-type strain continued to grow slowly for an additional 2 h. The viability of both strains was strongly influenced by heat shock. After 8 h, it was considerably lower compared to unstressed cultures and the difference was more dramatic after prolonged heat shock (Table 1). However, the deleterious effect of heat shock on survival of B. flavum BD2 was significantly higher (about 10-fold). Also, shift down (30°C to 10°C) of the temperature of growing cultures of B. flavum wild-type and sigB mutant significantly influenced the growth of both cultures. However, while the growth of wild-type continued at a reduced rate, the growth of B. flavum BD2 stopped almost immediately (Fig. 2F). However, the cold shock had only moderate effect on the viability of both cultures. Survival of both strains was comparable for both 8 h and 26 h stressed cultures (Table 1). As the growth medium LB used in the experiments is a poor medium for C. glutamicum, the data obtained in this study might only be applicable to a growth-limited culture.

Table 1.  Effect of the sigB mutation on the ability of B. flavum to survive various environmental stresses
  1. 14 h grown cultures were exposed to various stress conditions for 8 or 24 h. Survival is expressed as percentage of the viability in comparison to parallel unstressed culture at the same time point. The values shown are means±standard deviations.

Stress conditionsBacterial strain8 h stressed cells26 h stressed cells
pH 4.4B. flavum CCM 25135.2±5.814.6±3.7
 B. flavum BD210.8±2.82.3±0.4
39°CB. flavum CCM 25160.1±5.128.4±4.3
 B. flavum BD223.2±3.22.8±0.5
8% (v/v) ethanolB. flavum CCM 25156.3±5.521.1±5.7
 B. flavum BD212.4±2.62.7±0.8
10°CB. flavum CCM 25171.9±6.765.4±6.1
 B. flavum BD273.2±7.850.1±4.8
5% (w/v) NaClB. flavum CCM 25195.1±4.888.2±6.4
 B. flavum BD259.2±6.449.9±5.7
30 mM H2O2B. flavum CCM 25193.6±5.294.3±9.7
 B. flavum BD295.3±4.796.1±8.2

Though likely also B. flavum sigB forms a monocistronic transcription unit as its counterpart in B. lactofermentum[5,16], we wanted to know whether the B. flavum sigB mutation does not have any polar effect. We have complemented the mutant in trans with the wild-type B. flavum sigB gene including its promoter region (Section 2). The sigB mutant strain containing the corresponding plasmid pJUPB1 complemented all the phenotypic defects of the sigB mutant (data not shown).

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

During nutrient starvation and environmental stresses, bacteria induce sophisticated response mechanisms, which allow continued growth or survival under adverse conditions. In Gram-negative bacteria (such as E. coli, Salmonella typhimurium, and Yersinia spp.), the group 2 sigma factor σS has been shown to be a major regulator involved in starvation and stress response[3]. Until recently, no functional homolog of σS has been found in Gram-positive bacteria. In contrast, the group 3 alternative sigma factor σB is responsible for responses to both stimuli in B. subtilis[4]. Its functional homologs have been identified also in other Gram-positive bacteria, like Staphylococcus aureus and Listeria monocytogenes[4]. However, in S. aureus, it controls only environmental stress response, but not starvation survival[17]. Therefore, it has been suggested that σS and σB have parallel roles in the general stress response of Gram-negative and Gram-positive bacteria, respectively.

We have previously identified a homolog of primary sigma factor, SigB, in B. flavum, which belonged to the group 2 sigma factors[5]. Expression of the sigB gene was substantially increased after several stress conditions, suggesting its role in stress response[5]. In the present study, results of the phenotypic analysis of the B. flavum sigB mutant clearly confirmed this suggestion. B. flavum SigB has a role in response to several stress conditions, including acid, salt, ethanol, heat, and cold stresses. The growth of the B. flavum sigB mutant was most dramatically affected after acid, salt, and cold stresses (Fig. 2). Likewise, expression of B. flavum sigB is most responsive after acid and cold stresses[5]. Based on these results, B. flavum SigB seemed to be functionally most similar to L. monocytogenes stress response σB, which has a dominant role in osmotolerance, acid tolerance and growth at low temperature [18–20]. In addition to dramatic growth defect, the B. flavum sigB mutant failed to neutralize acid pH to physiological level after acidification, suggesting a role of SigB in the regulation of the process of adjustment of the pH. Salt stress was the other condition dramatically influencing growth rate of the sigB mutant (Fig. 2B). A protective system based on glycine betaine uptake, to overcome osmotic stress, was described in the close related bacterium C. glutamicum. This transport system was constitutively expressed at a basal level, but it was induced 8-fold by osmotic stress[21]. Likely, SigB might have a role in regulation of similar protective system in B. flavum. Similarly, in L. monocytogenes, a null mutation in the sigB gene, encoding stress response alternative sigma factor σB, led to substantial defects in its ability to use betaine and carnitine as osmoprotectants[18]. Cold shock is the next stress that most strongly affected growth of B. flavum sigB mutant (Fig. 2F). The critical role of sigma factor σB in adaptation to low temperature was also described for L. monocytogenes. In this organism, σB is necessary for efficient accumulation of betaine and carnitine as cryoprotectants[19]. As mentioned above, a betaine transport system was described also for corynebacteria[21], and a question has risen about its possible role in cold shock response. Interestingly, SigB seemed to have no role in oxidative stress response and stationary phase survival. Moreover, B. flavum was much more resistant to H2O2 compared to other Gram-positive and Gram-negative bacteria. The concentration as high as 30 mM had almost no effect on the growth of either B. flavum wild-type or sigB-disrupted strains (Fig. 2A); in contrast, about 5 mM H2O2 substantially inhibits growth of other bacteria (most dramatically in sigB-, or rpoS-disrupted strains). These results are partially consistent with the induction of sigB expression under these conditions, where the level of sigB transcript increased after salt, ethanol, acid, and cold stresses, however was not induced during stationary phase or after oxidative stress[5]. Interestingly, phenotypic analysis of sigB mutant suggests a role of B. flavum SigB in the heat shock response, no elevated level of the transcript after temperature shift to 45°C was indicated. On the contrary, the level of the sigB transcript was lowered, similarly as for the primary sigma factor gene sigA[5]. Likely, the shift to the temperature of 45°C was lethal for B. flavum, and an increased synthesis of mRNA could not occur. Another interesting feature of the B. flavum sigB mutant is its growth defect that suggests a participation of SigB also in regulation of some essential genes necessary for vegetative growth.

The function of SigB in B. flavum seems to be similar to that of general stress response σB from Gram-positive bacteria, such as B. subtilis, L. monocytogenes and S. aureus. However, they all belong to group 3 alternative sigma factors. As B. flavum SigB belongs to the group 2 nonessential homologs of primary sigma factors[5], it seems to be a functional homolog of general stress response σS of Gram-negative bacteria. A close homolog of B. flavum SigB has been identified in another representative of taxonomic order Actinomycetales, Mycobacterium tuberculosis. This sigma factor, SigB (MysB), whose amino acid sequence is 74% identical to B. flavum SigB, belongs also to group 2[22]. M. tuberculosis SigB has also been suggested to be involved in stress response, however, in contrast to B. flavum SigB, expression of its gene is induced preferentially in stationary phase, and after oxidative and heat stresses[23]. Moreover, M. tuberculosis SigB has been inferred to be involved in oxidative stress response, as it has a role in expression of the katG gene encoding hyperoxidase I[24]. Therefore, in spite of their high sequence similarities, stress response sigma factors SigB of M. tuberculosis and B. flavum are functionally distinct, perhaps related to their different habitats. B. flavum is a soil organism and M. tuberculosis an intracellular pathogen, and as such, it is exposed to oxidative stress environment of the host macrophage after infection. Likely, the response to hydrogen peroxide stress in corynebacteria is regulated through another pathway, not requiring the sigma factor SigB.

Interestingly, the B. flavum sigB gene is transcribed from a promoter (TGGGAACTT-N15-CGTTGAA-N6-G)[5], sequence of which is almost identical to M. tuberculosis sigB promoter (TGGGAACTC-N15-CGTTAAA-N6-G)[25]. In M. tuberculosis, the sigB promoter is under the overlapping control of two ECF sigma factors, SigE and SigH, that have been shown to have critical roles in the response to oxidative and heat stresses and survival in macrophages [25,26]. On the basis of the high sequence similarity of conserved −10 and −35 sequences of the sigB promoters in both organisms, we assume that also B. flavum sigB is under the control of an ECF sigma factor. Likely, a homolog of recently found putative ECF sigma factor σE from C. glutamicum (GenBank Acc. no. AF216691) might be a candidate for this recognition, assuming that it is present also in the closely related strain B. flavum.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

This work was supported by Grants 2/7001/22 and 2/2058/22 from Slovak Academy of Sciences.

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  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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