Discovery of a novel gene involved in autolysis of Clostridium cells

Abstract

Cell autolysis plays important physiological roles in the life cycle of clostridial cells. Understanding the genetic basis of the autolysis phenomenon of pathogenic Clostridium or solvent producing Clostridium cells might provide new insights into this important species. Genes that might be involved in autolysis of Clostridium acetobutylicum, a model clostridial species, were investigated in this study. Twelve putative autolysin genes were predicted in C. acetobutylicum DSM 1731 genome through bioinformatics analysis. Of these 12 genes, gene SMB_G3117 was selected for testing the intracellular autolysin activity, growth profile, viable cell numbers, and cellular morphology. We found that overexpression of SMB_G3117 gene led to earlier ceased growth, significantly increased number of dead cells, and clear electrolucent cavities, while disruption of SMB_G3117 gene exhibited remarkably reduced intracellular autolysin activity. These results indicate that SMB_G3117 is a novel gene involved in cellular autolysis of C. acetobutylicum.

Graphical abstract

Keywords

cell autolysis / autolysins / Clostridium / gene SMB_G3117

INTRODUCTION

Cell autolysis phenomenon is widespread among bacteria that possess peptidoglycan (Smith et al., 2000). Considering the possibility that cell autolysis is involved in selective removal of peptidoglycan, it might be involved in numerous cellular processes including cell growth, cell wall turnover, peptidoglycan maturation, cell division, cell lysis, motility, chemotaxis, genetic competence, differentiation, and pathogenicity (Foster, 1994; Blackman et al., 1998). Clostridium is one of the largest bacterial genera, ranking the second in size next to Streptomyces, and classified as Gram-positive endospore-forming obligate anaerobes (Andreesen et al., 1989; Rehner and Samuels, 1994). Many species of Clostridium are pathogenic, e.g. C. botulinum, C. septicum, and C. difficile, while some species of Clostridium, e.g. C. acetobutylicum and C. beijerinckii, are also of biotechnological importance for solvents production. Understanding the genetic basis of autolysis of Clostridium cells is thus important for obtaining insights into the life cycle of this important species.

Acetone-butanol-ethanol (ABE) fermentation by solvent-producing Clostridium represents one of the oldest industrial fermentation processes ever known, ranking second in scale only to ethanol fermentation by yeast (Lutke-Eversloh and Bahl, 2011). Recently, this process has been revived because of the potential application of butanol as an alternative biofuel (Lee et al., 2008). Typical batch fermentation process of C acetobutylicum can be divided into two phases, acidogenesis and solventogenesis. Exponentially growing cells mainly produce acids in acidogenic phase, which were then reassimilated and converted to solvents by late-exponential growing cells in solventogenic phase. In the latter stage of solventogenesis, the optical density (OD₆₀₀) value usually sharply decreases, the so-called autolysis (Barber et al., 1979; Allcock et al., 1981). Autolysis of Clostridium cells might account for the diminished and even ceased production of solvents.

It is generally accepted that autolysis is triggered by autolysins, which are a group of bacteriolytic enzymes that digest the bacterial cell-wall peptidoglycan (Shockman and Holtje, 1994; Smith et al., 2000). In Gram-positive bacteria, autolysins can be classified into four main categories according to the specificity of their hydrolytic bonds, i.e. muramidases, glucosaminindases, N-acetylmuramoyl-L-alanine amidases (amidases), and endopeptidases (Smith et al., 2000). In the last decades, autolysins in Escherichia coli (Holtje, 1995; Heidrich et al., 2001), Streptococcus (Ju et al., 2012; Tamura et al., 2012), Staphylococcus aureus (Jayaswal et al., 1990; Foster, 1995), lactic acid bacteria (ChapotChartier, 1996), and Bacillus subtilis (Blackman et al., 1998; Smith et al., 2000) have been extensively investigated. The autolysis phenomenon in ABE fermentation was firstly described in 1979 (Barber et al., 1979), but the molecular basis for autolysis of C. acetobutylicum remains poorly understood. Currently, only four lytic enzymes in solvent-producing clostridia have been partially purified and characterized. These include glycoprotein (28 kDa) (Webster et al., 1981), muramidase (41 kDa) (Croux et al., 1992b), and amidase (115 kDa) (Garcia et al., 1988) in C. acetobutylicum ATCC 824, and N-acetylmuramidase (44 kDa) in C. saccharoperbutylacetonicum N l-4 (Yoshino et al., 1982). A gene CA_C0554 in C. acetobutylicum ATCC 824, which encodes a lysozyme consisted of 324 amino acids with a calculated molecular mass of 34.939 kDa, was expressed in E. coli and characterized (Croux and Garcia, 1992).

The aim of this study was to identify and characterize genes that might be involved in cell autolysis of C. acetobutylicum DSM 1731. Autolysin relevant features including intracellular autolysin activity, growth profile, viable cell counts, and cellular morphology were performed. Finally, a novel gene, SMB_G3117, was found to play a role in the autolysis of C. acetobutylicum cells.

RESULTS

Identification of putative autolysin genes in C. acetobutylicum through bioinformatics analysis

There are comprehensive descriptions concerning autolysins in Bacillus subtilis (Smith et al., 2000), which is moderately related to C. acetobutylicum (Nolling et al., 2001), thus providing a theoretical reference for our research. In B. subtilis 168, eight genes representing conserved elements of four autolysin families (Table 1) were extracted from Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/). To identify genes encoding putative autolysin in C. acetobutylicum DSM 1731 (Bao et al., 2011), the sequences of 8 autolysin genes in B. subtilis 168 were individually used as query sequences to blast against C. acetobutylicum DSM 1731 genome with E-value < 10^-5. Nine homologous genes were identified in the genome of C. acetobutylicum DSM 1731. To avoid missing target genes relevant to autolysin in DSM 1731, the sequences of these 9 genes were used to identify other possible autolysin genes through function domains search, resulting in 3 more candidate genes identified (Table 1), in which SMB_G0566 and SMB_G1901 are paralog genes of SMB_G1126, while SMB_G3128 is paralog gene of SMB_G3117. In total, 12 putative autolysin genes were identified in the C. acetobutylicum DSM 1731 genome (Fig. 1). These genes can be divided into four categories on the basis of sequence similarities, i.e., muramidases (SMB_G0566, SMB_G1126, SMB_G1901), glucosaminindases (SMB_G2359), N-acetylmuramoyl-L-alanine amidases (SMB_G0700, SMB_G3117, SMB_G3128), and endopeptidases (SMB_G0078, SMB_G0429, SMB_G0493, SMB_G2513, SMB_G3064).

Preliminary evaluation of the putative autolysin genes in C. acetobutylicum

All 12 putative autolysin genes were overexpressed in C. acetobutylicum SMB009 which is capable of accepting unmethylated DNA (Dong et al., 2010) to evaluate whether these genes are involved in autolysis. Considering that constitutive expression of autolysin genes may lead to cellular toxicity, the inducible expression vector pGusA2-2tetO1 that we previously developed (Dong et al., 2012) was used.

Twelve isolated transformants were cultivated in CGM medium supplemented with 50 μg/mL erythromycin (Em), and 100 ng/mL anhydrotetracycline (aTc) for inducible expression. The results (Fig. 2A) showed that only inducible expression of gene SMB_G2359 (Fig. 2A-a) and SMB_G3117 (Fig. 2A-b) led to a 53.4% and 72.3% decrease of OD₆₀₀, respectively, as compared to that of the control. We also observed that inducible expression of SMB_G2359 and SMB_G3117 at OD₆₀₀ = 0.5 (around 6 h) (Fig. 2B) led to a 13% and 10% decrease of OD₆₀₀, while the non-induced strain (Fig. 2C) showed the same OD₆₀₀, as compared to the control. This suggests that gene SMB_G2359 and SMB_G3117 might play a role in cell lysis.

To investigate the specific function of the selected gene SMB_G2359 and SMB_G3117, attempts to disrupt these genes in C. acetobutylicum DSM1731 were made using ClosTron pMTL008 vector (Dong et al., 2010; Heap et al., 2010). To disrupt gene SMB_G2359, three targeted intron insertion sites (732/733s, 867/868s, and 1292/1293s) were tested, respectively. For each target site, more than 200 integrants were screened through colony PCR (Table S1). Unexpectedly, no positive integrants were obtained, suggesting that gene SMB_G2359 might be essential for cell growth. Gene SMB_G3117 was successfully disrupted, generating mutant strain KO3117. Therefore, gene SMB_G3117 was selected for further investigation.

Functional analysis of gene SMB_G3117 in C. acetobutylicum DSM 1731

To further investigate the function of gene SMB_G3117, we compared the autolysis related features of the SMB_G3117 disrupted strain KO3117, the SMB_G3117 inducibly expressed strain DSM 1731 (p3117), with their control strains wild type strain DSM 1731 and strain DSM 1731 (p2tetO1), respectively. The tested features include intracellular autolysin activity characterized as the decrease of OD₆₀₀ in sodium phosphate buffer, growth profile (increase of OD₆₀₀), viable cell counts (spreading plates and colony counting), and cellular morphology (TEM observation).

Compared to wild type strain DSM 1731, the intracellular autolysin activity of the mutant strain KO3117 decreased by 37.7% (Fig. 3C). Nevertheless, no significant difference was observed in growth profile, viable cell numbers (Fig. 3A and 3B), and cellular morphology (Fig. 4C and 4D) between these two strains. We examined the dynamic transcriptional profile of C. acetobutylicum ATCC 824 published in a previous study (Jones et al., 2008) and calculated the expression level of relevant genes. The expression level of the gene CA_C3081 in strain ATCC 824 (same to SMB_G3117 in strain DSM 1731; the genome of strain ATCC 824 and DSM 1731 are highly similar (Bao et al., 2011)) was 5-fold lower than the average transcription level of all genes during vegetative growth (Fig. 5). This suggests that the expression level of SMB_G3117 might be quite low, so that the disruption of this gene in wild type did not exhibit clear phenotype.

Clear autolysis related phenotypes were observed in strain DSM 1731 (p3117). Growth profile assay also revealed that the cease of growth of strain DSM 1731 (p3117) occurred at 12 h, which was 12 h ahead of the control strain (Fig. 3D). In addition, the viable cell numbers of strain DSM 1731 (p3117) were 15-fold less than its control strain (Fig. 3E). Moreover, TEM analysis (Fig. 4A and 4B) showed that electrolucent cavities, which were termed as “nuclear vacuoles” previously (Eltsov and Zuber, 2006), were clearly observed in strain DSM 1731 (p3117) at 12 h, in sharp contrast with that of the control strain. We also observed that the intracellular autolysin activity of strain DSM 1731 (p3117) was increased by 14.0% (Fig. 3F), not as high as expected though. All these observations conceive that SMB_G3117 overexpression led to significant cellular autolysis during vegetative growth.

DISCUSSION

In this study, two genes SMB_G2359 and SMB_G3117 that might contribute to autolysis of C. acetobutylicum were identified from twelve putative autolysin genes under the available experimental conditions. Gene SMB_G2359, annotated as N-acetylglucosaminidase domain- and ChW repeat-containing cell wall hydrolase (encoded by lytD gene), is the only predicted autolysin gene in LytD glucosaminidase family. In B. subtilis, only one lytD gene was identified and proved to be one of the major autolysins. However, the lytD mutant of B. subtilis still exhibits glucosaminidase activity, suggesting alternative glucosaminidase genes may exist in the genome (Rashid et al., 1993). The failure to disrupt SMB_G2359 in this work may indicate that this gene is the only one responsible for glucosaminidase activity in C. acetobutylicum.

Inducible expression of gene SMB_G3117 led to severely weakened cell viability and integrity, and caused cell autolysis. However, disruption of this gene in C. acetobutylicum DSM1731 did not exhibit growth difference compared to wild type strain. This suggests that the expression of gene SMB_G3117 is tightly regulated to avoid overexpression to a lethal level. Although gene SMB_G3117 is annotated as spore-cortex-lytic enzyme, a type of N-acetylmuramoyl-L-alanine amidases, our results showed that overexpression of gene SMB_G3117 triggered autolysis of C. acetobutylicum during vegetative growth. In another recent study, a novel Streptococcus suis gene atl containing one N-acetyl-muramoyl-L-alanine amidase domain, also exhibited autolysis activity in conditions consistent with our study, and was proved to take part in cell autolysis and separation of daughter cells (Ju et al., 2012). This suggests that the autolysis function of SMB_G3117, as revealed in this study, might be an important part of the life cycle of C. acetobutylicum cells that is worthy of further investigation.

MATERIALS AND METHODS

Strains, plasmids and culture conditions

The bacterial strains and plasmids used in the present study are listed in Table 2. Escherichia coli strains were routinely grown aerobically at 37°C and 200 r/min in liquid LB medium or solid LB with agar (1.5%) medium supplemented, when necessary, with ampicillin (100 mg/mL) and/or chloramphenicol (Cm, 30 mg/mL). All C. acetobutylicum strains were grown anaerobically at 37°C in liquid RCM medium (Hirsch and Grinsted, 1954) or solid RCM with agar (1.5%) medium supplemented, when necessary, with chloramphenicol (Cm, 30 mg/mL), erythromycin (Em, 50 mg/mL) and anhydrotetracycline (aTc, 100 ng/mL). The CGM medium (Hartmanis and Gatenbeck, 1984) was also used for C. acetobutylicum growth. All C. acetobutylicum and E. coli strains were maintained frozen in 15% (v/v) glycerol at –80°C.

Construction of gene SMB_G3117 inducible expression strains

The primers used to amplify these 12 genes are listed in Table S1. Twelve inducible expression vectors were constructed by replacing gusA gene with corresponding putative autolysin genes in pGusA2-2tetO1 using restriction enzyme SpeI (or NcoI) and XhoI. The control plasmid p2tetO1 was generated by removing gusA gene in pGusA2-2tetO1 using the same restriction enzyme digestion, end-filling using high fidelity DNA polymerase, and then self-ligation. Inducible-autolysin-expression strains were obtained by electrotransforming the respective 12 plasmids into C. acetobutylicum SMB009 according to the protocol developed previously (Mermelstein et al., 1992).

Construction of gene SMB_G3117 disrupted mutant

Retargeted gene ClosTron plasmids were constructed as previously described using plasmid pMTL008 (Dong et al., 2010). The primers used for the construction of these plasmids are listed in Table S1. The resulted plasmid, designated as pMTL008-3117, was confirmed by DNA sequencing using primer 14-007-R1. pMTL008-3117, methylated by co-transforming pAN2 into E. coli TOP10, was then electrotransformed into the strain DSM1731, resulting in strain KO3117. Integrants were selected on the basis of acquisition of chloramphenicol resistance, and then reselected again through erythromycin resistance. The positive integrant was screened through a number of diagnostic PCR and further confirmed by three pairs of primers (Fig. S1). Finally, the fragments generated were subject to nucleotide sequence analysis to definitively confirm that insertion had occurred at the desired position.

Growth measurement

Cell optical densities at 600 nm were measured dynamically using a UV-visible spectrophotometer (UV-2802PC, Unico, Shanghai, China). Samples were diluted in the appropriate medium to ensure an absorbance below 0.50.

Viable cell counts assay

A CFU (colony-forming unit) assay is employed to count the viable cell numbers (Zingaro and Terry Papoutsakis, 2012). The unit of measurement is CFU/mL. Strains were cultivated in CGM medium supplemented with 50 μg/mL erythromycin (Em), and 100 ng/mL aTc for inducible expression. Samples from different growth phases were serially diluted in sterile liquid media. Dilution level was determined based on A₆₀₀ measurements of the samples. One hundred microliter diluted broth was taken from dilution series and spread onto agar solidified CGM plate containing appropriate antibiotics. Viable cell numbers were calculated after incubating anaerobically at 37°C for 24 h.

Intracellular autolysin activity of the inducible expression and disruption of gene SMB_G3117

Intracellular autolysin activity assay of the inducible-expression and disruption of gene SMB_G3117 was performed according to modified procedures (Croux et al., 1992a). Exponential phase cells (OD₆₀₀ = 2.0) were harvested by centrifugation (12,000 g, 5 min, 4°C) and after the supernatant fluid had been discarded, residual growth medium was removed with cotton tips. The pellet was washed once with 0.1 mol/L sodium phosphate buffer (pH 6.3). The washed pellet was then diluted to an initial OD₆₀₀ of 1 into preincubated (37°C) 0.1 mol/L sodium phosphate buffer (pH 6.3). Note that 100 ng/mL aTc was added in the inducible-expression group. The decreases in OD₆₀₀ were monitored at 37°C every 0.5 h.

Cellular morphology of the inducible expression and disruption of gene SMB_G3117 through TEM observation

All strains were cultivated as mentioned in growth and viable cell counts assay. Cells were collected by centrifugation (12,000 g, 5 min, 4°C) at different phase of growth (0 h, 12 h, 24 h, 48 h) then fixed instantly by adding 2.5% (v/v) glutaraldehyde for more than 2 h, respectively. Samples for TEM were prepared as described previously (Jones et al., 2008; Zhang et al., 2010). Prepared cell thin sections were examined with a transmission electron microscope (JEM-1400; JEOL Ltd., Japan) operating at an acceleration voltage of 80 kV.

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