Introduction

A biofilm is multicellular communities in which cells adhere to each other or solid surface. These adhering cells are frequently encapsulated within a self-produced matrix of extracellular polymeric substance. Biofilm cells significantly enhance their resistance to antibiotics and the human immune system than their planktonic counterparts. Biofilm formation is initiated with surface attachment of planktonic bacteria, followed by formation of clusters and microcolonies, and subsequent development of differentiated structures in which individual bacteria and the entire community are enclosed by exopolysaccharides. And when biofilms disperses, the biofilm development cycle comes full circle [17].

While the ability of Mycobacterium tuberculosis to form biofilms remains obscure, many other species of mycobacteria, including M. avium, M. fortuitum, M. marinum, and M. smegmatis, are well-documented biofilm producers. However, little is known about the molecular mechanisms involved in mycobacterial biofilm formation, the nature of the matrix or the biofilm structure. Previous articles reported that M. smegmatis forms biofilms involving in glycopeptidolipids [26, 27] and mycolyl-diacylglycerols [6], and the amphiphilic molecules model elucidated the function of these molecules in biofilm formation [27]. Additionally, some other factors such as undecaprenyl phosphokinase [30], serine/threonine protein kinase [9], iron [19, 41], and GroEL1 are required for biofilm formation. M. smegmatis biofilm formation earlier events involve attachment and spreading, followed by the maturation and matrix formation. Because mycobacterial genomes do not contain the genetic material for exopolysaccharide biosynthesis [41], the matrix is probably primarily composed of fatty acids. For example, GroEL1 is required for fatty acid synthesis, which results in the increased synthesis of C56–C68 fatty acids during the biofilm formation, and a mutant defective in GroEL1 is specifically deficient in the late stages of biofilm formation [18]. Besides, the availability of iron for M. smegmatis biofilm maturation correlates closely with the synthesis of C56–C68 fatty acids [19, 41].

Due to mycobacteria being nonflagellated microorganisms, sliding motility is produced by the expansive forces of the growing bacterial population and cell surface properties that favor reduced friction between the cells and substrate, and the result is the slow movement of a uniform monolayer of cells as a unit [14]. Both non-pathogenic M. smegmatis and the opportunistic pathogen M. avium are able to slide, and this sliding motility correlates with the presence of glycopeptidolipids and mycolyl-diacylglycerols [27]. So the ability of mycobacteria to slide over the surface of motility plates is relative with biofilm formation.

Inorganic polyphosphate (polyP) is a linear polymer of hundreds of orthophosphate (Pi) residues linked by ATP-like, high-energy, phosphoanhydride bonds and found in all organisms [21]. In bacteria, two main kinds of enzymes are involved in the metabolism of polyP: polyphosphate kinases (PPK1 and PPK2) catalyze the reversible conversion of the terminal phosphate of ATP (or GTP) into polyP; exopolyphosphatase (PPX) progressively hydrolyzes the terminal residues of polyP to liberate Pi [2, 12]. Studies with ppk1 bacterial mutants have showed that polyP function might be promiscuous, such as inhibition of RNA degradation, activation of Lon protease during stringent response, engagement in membrane channel structure [28, 29], correlated with the resistance to diverse stresses such as heat, oxidants, osmotic challenge, antibiotics and UV, and influence on motility, biofilm development, quorum sensing, sporulation, and virulence [23–25, 31]. Particularly, polyP is also involved in the M. tuberculosis macrophage survival [34, 35].

However, previous experiments with ppk1 bacterial mutants might be flawed by a neglect of endogenous polyP fluctuation. For example, the stationary phase Escherichia coli ppk mutant was reportedly susceptible to oxidant [7], heat shock, and osmotic stress [22]. However, oxidant resistant visible small-colonies occasionally emerged from the stationary cultures of ppk mutant [22]. One possible cause for these emerging papillate colonies might be the short chain polyP produced by the ppk mutant via an alternative pathway [3]. To preclude the endogenous polyP interference, the introduction of recombinant exopolyphosphatase is advisable [4, 32]. This methodology was pursued in our investigation into the role of PolyP in mycobacterial biofilm and colony morphology. We speculate that two M. tuberculosis genome annotation of conserved hypothetical protein homologs of PPX1 (Rv1026) and PPX2 (Rv1026) might be functional exopolyphosphatases. A structure-based sequence comparison of PPX [13] revealed that the PPX of M. tuberculosis have biological activity. As the first attempt, Rv1026 was cloned into the pMV261, which harbor an expression cassette containing 404 bp of the 5′ regulatory region of the BCG hsp60 gene and can induce the expression of its downstream gene even at 37°C [1, 33]. The resulting plasmid pMV261-Rv1026 (pRv1026) and the plasmid pMV261 were transformed into M. smegmatis strain mc2155 by electroporation. We found that the M. smegmatis (pRV1026) showed relatively decreased polyP concentration, and displayed a phenotype that was different from the M. smegmatis (pMV261) in sliding motility and biofilm formation, the surfactant Tween 80 enhancing this effect on the sliding motility and biofilm formation of the M. smegmatis (pRV1026). Comparison of the fatty acid composition of the M. smegmatis (pRV1026) with the M. smegmatis (pMV261) revealed that there are four different peaks between both strains. These results indicate that polyP can affect the fatty acid composition of cell wall and these alteration can alter the strains ability to slide and form biofilm.

Materials and Methods

Bacterium Strains and Growth Conditions

Mycobacterium smegmatis strain mc2155 was routinely grown in Middlebrook 7H9 broth with 0.05% Tween 80 unless otherwise indicated and plated in the Middlebrook 7H10 supplemented with 1% glucose and 20 μg/ml of kanamycin unless otherwise indicated.

Molecular Cloning and Electroporation

The open reading frame of Rv1026 of M. tuberculosis was PCR amplified using the forward primer 5′-CTGGTGGATCCGCAGTGG-3′ and the reverse primer 5′-CGGGCAGAAGCTTGTCCC-3′. The resulting PCR product was digested with bamHI and HindШ and cloned into pMV261 pretreated with the same enzymes to yield pRv261.

The plasmids pRv1026 and pMV261 were introduced into M. smegmatis mc2 155 by electroporation.

In Vitro Growth Kinetics and the Concentration of polyP of M. smegmatis Strains

Fresh mid log-phase cultures of the M. smegmatis (pRV1026) and the M. smegmatis (pMV261) were inoculated in M7H9 supplemented with 0.05% Tween80 to an initial OD600 of 0.02. Aliquots of 2 ml were removed at suitable intervals up to 29 h and growth was monitored by measuring OD600 values. After the measure of OD600 values, the aliquots of 2 ml were centrifuged with 12,000 r/min for 1 min and the cell pellet was used to assay total intracellular polyP by a modification of the methods of Werner, TP et al. [39], and McGrath, JW and Quinn, JP [15]. Briefly, the cell pellet was extracted for maximal 5 min at room temperature after resuspending the cell in 50 μl 1 M sulfuric acid. The suspension was neutralized with 50 μl of 2 M NaOH. Cell fragments were removed by centrifugation. To determine total intracellular polyP, 100 μl of concentrated HCl was added to 0.5 ml cell extract and heated at 100°C for 45 min; the phosphate liberated was assayed by the method of Werner, TP et al. [39]. 86 μl of 28 mM ammonium heptamolybdate in 2.1 M H2SO4 and 64 μl of 0.76 mM malachite green in 0.35% polyvinyl alcohol were added. The OD595 was measured in a SpectraMax190 produced by molecular device. The polyP concentrations were expressed in OD595 and are given as means of triplicates.

Sliding Motility of Mycobacteria

The M. smegmatis (pRv1026) and M. smegmatis (pMV261) grown on M7H9 broth (with 1% glucose, 0.05% Tween 80, and 20 μg/ml of kanamycin) were inoculated via sterile toothpicks onto M7H9 plates containing 0.3% agarose without any carbon source with/without 0.05% Tween 80. The plates were incubated at 37°C for 4–5 days.

Determination of Biofilm Formation

M. smegmatis strains were inoculated into 96-well flat bottom plate which every cell filled with 1 ml of M7H9 medium supplement with/without 0.05% Tween 80. Then, cells were incubated at 37°C without disturbance for 4–5 days. The M. smegmatis (pRv1026) and M. smegmatis (pMV261) were grown in 96-well plates, stained with 1% crystal violet and assayed for biofilm formation by spectrophotometric reading of the ethanol extract at 570 [5, 19]. Biofilm formation was analyzed by growing the strains in M7H9 media without Tween 80 and with 0.05% Tween 80 in triplicate, respectively, in 96-well plate. Cells were stained with 1% crystal violet, washed with water and crystal violet dissolved using 95% ethanol.

Extraction of Fatty Acid of Mycobacteria and Gas Chromatography Analysis

Lipid extracts from cells and gas chromatography analysis are accomplished following instruction of the Microbial Identification System (version 6.0).

Results and Discussion

The Intracellular polyP Concentration and In Vitro Growth Kinetics of the M. smegmatis

The M. smegmatis (pRv1026) showed reductive intracellular polyP concentration compared with M. smegmatis (pMV261) at early stationary phases, indicating that Rv1026 affects the polyP concentration of stationary phases cell (Fig. 1). However, the M. smegmatis (pRv1026) showed higher polyP concentration in exponential phase compared with the M. smegmatis (pMV261). This phenomenon maybe caused by the polyphoshate kinases (PPK1 and PPK2), which catalyze the reversible conversion of the terminal phosphate of ATP (or GTP) into polyP. Recently, Jagannathan et al. reported that the reversibility of the enzymatic activity of PPK1 may tilt the balance of the reaction one way or the other [11]. They speculated that the forward reaction of PPK1 is predominantly operative during the log phase, and result in polyphosphate production; during the stationary phase, the reversal reaction is triggered to synthesize ATP [11]. The existence of PPK1 and PPK2 in M. smegmatis genome suggests that the M. smegmatis PPK might be functional. So the phenomenon that the concentration of polyP in M. smegmatis (pRv1026) during exponential phase is not reductive, but increasing, may indicate that there are sufficient nutrition to supply PPK with ATP to synthetize polyP. However, during stationary phase, the polyP concentration in M. smegmatis (pRv1026) is more reductive than that in M. smegmatis (pMV261), because not only the reverse reaction of PPK is operative to synthesize ATP, but also the Rv1026 hydrolyzes the terminal residues of polyP to liberate Pi.

Fig. 1
figure 1

Intracellular polyP concentration in M. smetmatis mc2155 and the growth characteristics in vitro. The M. smegmatis (pRv1026) and the M. smegmatis (pMV261) were inoculated at a final OD600 of 0.05 into fresh medium. Aliquots of 2 ml were taken out from the M. smegmatis (pMV261) (filled diamond) and the M. smegmatis (pRv1026) (filled triangle) at time points indicated and measured for OD600. After the measure of OD600, the aliquots of 2 ml were used to extract polyP as described in method. The polyP concentration in the M. smegmatis (pRv1026) (black column) and the M. smegmatis (pMV261) (white column) was denoted as OD595 of Pi. Values plotted are the averages and SD from triplicate experiments. An unhydrolyzed sample was used as a control to determine the background level of Pi

Full size image

The reduction of polyP concentration in M. smegmatis (pRv1026) have no apparent effect to the planktonic growth (Fig. 1), which is consistent with the growth curves of ppk mutant of E. coli and Pseudomonas aeruginosa [24]. So we analyze the effect of reduction of polyP on sliding motility and morphology of the M. smegmatis (pRv1026) and M. smegmatis (pMV261).

Analysis of Sliding Motility and Morphology of M. smegmatis

Previous studies have shown that polyP-defective mutants like Paeruginosa [8, 23–25], Bacillus cereus [31], Dictyostelium discoideum [42], and Burkholderia pseudomallei [37] have defective motility including swimming motility, swarming and twitching motilities. In spite of being nonflagellated microorganisms, mycobacteria can spread on the surface of solid growth medium by a sliding mechanism [14]. This form of surface motility is produced by the expansive forces of the growing bacterial population and cell surface properties that favor reduced friction between the cells and substrate, and the result is the slow movement of a uniform monolayer of cells as a unit [10]. The lipid components of cell wall including glycopeptidolipids (GPLs) [26, 27] and mycolyl-diacylglycerols (MDAGs) [6] contribute to sliding motility and biofilm formation. We found that the M. smegmatis (pRv1026) showed morphological variations compared with M. smegmatis (pMV261) grown on 0.3% agarose plates without 0.05% Tween80 (Fig. 2a (1,2)) and with 0.05% Tween 80 (Fig. 2b (1,2)), respectively.

Fig. 2
figure 2

Morphology and sliding motility of M. smegmatis. a Morphology of M. smegmatis (pMV261) (1) and the M. smegmatis (pRv1026) (2) grown on 0.3% agarose plate without Tween 80. b Morphology of the M. smegmatis (pMV261) (1) and the M. smegmatis (pRv1026) (2) grown on 0.3% agarose plate with 0.05% Tween 80. c Sliding motility of the M. smegmatis (pMV261) (1) and the M. smegmatis (pRv1026) (2) grown on 0.3% agarose plate without Tween 80. d Sliding motility of the M. smegmatis (pMV261) (1) and the M. smegmatis (pRv1026) (2) grown on 0.3% agarose plate with 0.05% Tween 80

Full size image

Differences in colony morphology were also shown to be associated with the ability of sliding motility. The motility of the M. smegmatis (pRv1026) was slightly increased when compared with the M. smegmatis (pMV261) on 0.3% agarose plates without Tween80 (Fig. 2c (1,2)). In contrast, colony of the M. smegmatis (pRv1026) grown on 0.3% agarose plate with 0.05% Tween80 showed distinguished reduction in diameter as compared with M. smegmatis (pMV261) (Fig. 2d (1,2)), indicating that the sliding motility of the M. smegmatis (pRv1026) is more sensitive to denaturant such as Tween 80 than the M. smegmatis (pMV261). Previous article had reported that Tween 80 alters the cell surface properties and expose gradually more deeply buried lipids [20]. And recently, many evidences indicate that polyP is an active factor engaged in transcriptional regulation and metabolism [36, 38, 40]. So we hypothesize that the polyP can cause the variation of constituents of bacterial cell envelope by involvement in membrane channel structure or involvement in the transcriptional regulation of genes encoding lipids on the cell envelopes of mycobacteria.

The Altered Biofilm Formation of Recombinant

Since the M. smegmatis (pRv1026) forms drastically different colonies on agarose plates with 0.05% Tween80 from M. smegmatis (pMV261), we also examined that whether the ability of biofilm formation is different between the two strains. Using an assay to determine biofilm formation [5, 19], we found that the M. smegmatis (pRv1026) formed more biofilms in comparison with the M. smegmatis (pMV261) grown on media without Tween 80 (Fig. 3a). However, the M. smegmatis (pRv1026) formed less biofilms compared with the M. smegmatis (pMV261) on media with 0.05% Tween 80 (Fig. 3b). This result was also conformed by Quantitative estimation of crystal violet staining (Fig. 3c).

Fig. 3
figure 3

Biofilm formation associated with polyP depletion. a, b Analysis of biofilm formation. The dissolved crystal violet of the M. smegmatis (pMV261) (row A), the M. smegmatis (pRv1026) (row B) and M7H9 (no strains) (row C) grown without Tween 80 (a) and with 0.05% Tween 80 (b) was transferred to novel 96-well plate in triplicate columns 1, 2, and 3. c Estimation of crystal violet uptake. OD570 was measured to calculate the amount of crystal violet uptake by the M. smegmatis (pMV261) (A), the M. smegmatis (pRv1026) (B) and M7H9 media (C) grown without Tween 80 (black bar) and with 0.05% Tween 80 (blank bar). The experiment war carried out in triplicate and the graph indicates the mean value with standard deviation bars

Full size image

A previous study proposed a model for the role of GPLs in sliding motility and biofilm formation based on interaction between the mycobacterial cell surface and either hydrophilic (agarose) or hydrophobic (polystyrene) surfaces [27]. In addition, the loss of hydrophobic mycolyl-diacylalycerols (MDAGs) reduces the hydrophobicity of cell surface and biofilm formation [6]. Preliminary studies showed there are four different peaks between the gas chromatography of cellular wall fatty acid of the M. smegmatis (pRv1026) and the M. smegmatis (pMV261) (Fig. 4). These results indicate that polyP deficiency can affect the fatty acid composition of cellular wall. These alteration of cell wall might elucidate the reductive ability of strains to slide and form biofilm. Colony morphology is a complex phenotype influenced by the ability of cells to interact with one another. Cell wall architecture is not only responsible for distinctive colony morphology, but also contributes to virulence, persistence in macrophages, and modulation of the host immune response. Presently, the known lipoids including glycopeptidolipids and mycolyl-diacylglycerols affect mycobacterial phenotypes such as colony morphology, sliding motility, and biofilm formation. Additionally, the nutrition factor such as iron [19] and signal transduction such as two-component system [16] and serine/threonine protein kinase [9] also have effect on mycobacterial phenotypes. As to know, this is the first report on the functional analysis of a putative Rv1026 from M. tuberculosis and might provide new information about the role of polyP in affecting cell wall architecture. Further studies are needed to understand the mechanisms underlying the effect of polyP on cell wall architecture.

Fig. 4
figure 4

Gas chromatogram analysis of fatty acid. a The fatty acid profile of strain harboring pMV261. b The fatty acid profile of the recombinant. The arrowheads indicate the different peaks between strain harboring pMV261 and recombinant

Full size image