Cell wall metabolism in Bacillus subtilis subsp. niger: accumulation of wall polymers in the supernatant of chemostat cultures

Cell wall polymers were measured both in the cells and in the cell-free medium of samples from steady-state chemostat cultures of Bacillus subtilis, growing at various rates under magnesium or phosphate limitation. The presence of both peptidoglycan and anionic wall polymers in the culture supernatant showed the occurrence of wall turnover in these cultures. Variable proportions of the total peptidoglycan present in the culture samples were found outside the cells in duplicate cultures, indicating that the rate of peptidoglycan turnover is variable in B. subtilis. Besides peptidoglycan, anionic wall polymers were detected in the culture supernatant: teichoic acid in magnesium-limited cultures and teichuronic acid in phosphate-limited cultures. In several samples, the ratio between the peptidoglycan and the anionic polymer concentrations was significantly lower in the extracellular fluid than in the walls. This divergency was attributed to the occurrence of direct secretion of anionic polymers after their synthesis.     

Autolytic enzyme-deficient mutants of Bacillus subtilis 168.

Mutants of Bacillus subtilis strain 168 have been isolated that are at least 90 to 95% deficient in the autolytic enzymes N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase. These mutants grow at normal rates as very long chains of unseparated cells. The length of the chains is directly related to the growth rates. They are nonmotile and have no flagella, but otherwise appear to have normal cell morphology. Their walls are fully sysceptible to enzymes formed by the wild type and have the same chemical composition as the latter. Cell wall preparations from the mutants lyse at about 10% of the rate of those from the isogenic wild type, with the correspondingly small liberation of both the amino groups of alanine at pH 8.0 and of reducing groups at pH 5.6. Likewise, Microcococcus luteus walls at pH 5.6 and B. subtilis walls at pH 8 are lysed only very slowly by LiCl extracts made from the mutants as compared with rates obtained with wild-type extracts. Thus, the activity of both autolytic enzymes in the mutants is depressed. The frequencies of transformation, the isolation of revertants, and observations with a temperature-sensitive mutant all point to the likelihood that the pleiotropic, phenotypic properties of the strains are due to a single mutation. The mutants did not produce more protease or amylase than did the wild type. They sporulate and the spores germinate normally. The addition of antibiotics to exponentially growing cultures prevents wall synthesis but leads to less lysis than is obtained with the wild type. The bacteriophage PBSX can be induced in the mutants by treatment with mitomycin C.     

Effects of Mutational Loss of Specific Intracellular Proteases on the Sporulation of Bacillus subtilis

Two protease-deficient mutants of Bacillus subtilis 168 (Trp(-)) were isolated and compared with the parental strain with respect to production of intracellular proteases and sporulation. A mutant lacking the metal-requiring "neutral" protease intracellularly sporulated as well as the parental strain. A second mutant, deficient in an as yet uncharacterized intracellular protease, failed to sporulate normally. It is proposed that this new protease is also involved in intracellular protein turnover.     

Stabilization of cell wall proteins in Bacillus subtilis: a proteomic approach

Even though cell wall proteins of Bacillus subtilis are characterized by specific cell wall retention signals, some of these are also components of the extracellular proteome. In contrast to the majority of extracellular proteins, wall binding proteins disappeared from the extracellular proteome during the stationary phase and are subjected to proteolysis. Thus, the extracellular proteome of the multiple protease-deficient strain WB700 was analyzed which showed an increased stability of secreted WapA processing products during the stationary phase. In addition, stabilization of the WapA processing products was observed also in a sigD mutant strain which is impaired in motility and cell wall turnover. Next, we analyzed if proteins that can be extracted from B. subtilis cell walls are stabilized in the WB700 strain as well as in the sigD mutant. Thus, the cell wall proteome of B. subtilis wild type was defined showing most abundantly cell wall binding proteins (CWBPs) resulting from the WapA and WprA precursor processing. The inactivation of extracellular proteases as well as SigmaD caused an increase of CWBP105 and a decrease of CWBP62 in the cell wall proteome. We conclude that WapA processing products are substrates for the extracellular proteases which are stabilized in the absence of sigD due to an impaired cell wall turnover.     

Ribonuclease U2: cloning, production in Pichia pastoris and affinity chromatography purification of the active recombinant protein

A recombinant lipase, CWB-LipB, localized on the Bacillus subtilis cell surface and retaining lipase activity was unstable and not accumulated in a high yield. To improve the accumulation, we examined cell wall binding protease (wprA)- and/or sigma D (sigD)-deficient mutants, and also a NprE and AprA protease-deficient mutant as host strains. The nprE aprA mutation did not lead to a significant increase in the CWB-LipB accumulation. The wprA mutant accumulated a greater amount than the wild-type only in the stationary phase, but the sigD mutant accumulated a greater amount in both the exponential and stationary phases. The double mutant exhibited great accumulation of CWB-LipB, the amount being 36% of the total proteins extracted from the cell surface.     

Analysis of autolysins in temperature-sensitive morphological mutants of Bacillus subtilis.

The content and distribution of autolysin were measured in temperature-sensitive morphological mutants of Bacillus subtilis. Strains RUB1000 and RUB1012 grew as rods at 30 C. At 45 C the mutants contained disproportionately less teichoic acid than peptidoglycan and grew as irregular spheres. The amount of enzyme that could be extracted from rods was at least 31 times the amount extracted from spheres. The rate of autolysis of cell walls was 7- to 28-fold greater in rods than in spheres. The low activity found associated with the cell walls of spheres was not compensated for by larger amounts of autolytic activity in the cytoplasm. No activity was found in the growth medium at either temperature. The failure of the mutant cells to autolyze was due to low amidase activity and relatively resistant cell walls. Revertants of RUB1012 were isolated that had 13, 23, and 55% of the normal proportions of teichoic acid when grown at the nonpermissive temperature. Cell walls from the revertants were as sensitive to added amidase as the wild-type strain. None of the revertant strains regained the wild-type ability to produce more amidase at 45 C. However, the deficiency in autolysin observed with RUB1012 was partially restored in revertants containing higher proportions of teichoic acid.     

Teichoic acid is an essential polymer in Bacillus subtilis that is functionally distinct from teichuronic acid

Wall teichoic acids are anionic, phosphate-rich polymers linked to the peptidoglycan of gram-positive bacteria. In Bacillus subtilis, the predominant wall teichoic acid types are poly(glycerol phosphate) in strain 168 and poly(ribitol phosphate) in strain W23, and they are synthesized by the tag and tar gene products, respectively. Growing evidence suggests that wall teichoic acids are essential in B. subtilis; however, it is widely believed that teichoic acids are dispensable under phosphate-limiting conditions. In the work reported here, we carefully studied the dispensability of teichoic acid under phosphate-limiting conditions by constructing three new mutants. These strains, having precise deletions in tagB, tagF, and tarD, were dependent on xylose-inducible complementation from a distal locus (amyE) for growth. The tarD deletion interrupted poly(ribitol phosphate) synthesis in B. subtilis and represents a unique deletion of a tar gene. When teichoic acid biosynthetic proteins were depleted, the mutants showed a coccoid morphology and cell wall thickening. The new wall teichoic acid biogenesis mutants generated in this work and a previously reported tagD mutant were not viable under phosphate-limiting conditions in the absence of complementation. Cell wall analysis of B. subtilis grown under phosphate-limited conditions showed that teichoic acid contributed approximately one-third of the wall anionic content. These data suggest that wall teichoic acid has an essential function in B. subtilis that cannot be replaced by teichuronic acid.     

Relationship between lysostaphin endopeptidase production and cell wall composition in Staphylococcus staphylolyticus.

Mutants of Staphylococcus staphylolyticus incapable of producing an extracellular staphylolytic glycylglycine endopeptidase were isolated and found to have cells in the population susceptible to lysis by this enzyme, as did the wild-type organism under conditions in which the endopeptidase was not produced. These results suggest that cultures of this organism normally contain a heterogeneous population of cells with regard to cell wall composition and susceptibility to the enzyme. Production of the endopeptidase appears to act as a selective pressure which removes the susceptible cells in the population as the enzyme appears in the medium. A comparison of the peptidoglycan of the wild-type organism grown under conditions in which the endopeptidase was produced with that of this organism grown under nonproducing conditions and with those of endopeptidase-less mutants showed that in the presence of the endopeptidase the cell population had peptidoglycan with shorter peptide cross bridges and a greater percentage of serine in these cross bridges than was found in cells grown in the absence of the enzyme. The inability of the endopeptidase to hydrolyze glycylserine and serylglycine peptide bonds suggests that at least part of the resistance this organism has to the endopeptidase is due to relative amounts of serine found in the peptide cross bridges of some cells in the population.     

Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acid

Bacillus subtilis 168ts-200B is a temperature-sensitive mutant of B. subtilis 168 which grows as rods at 30 C but as irregular spheres at 45 C. Growth at the nonpermissive temperature resulted in a deficiency of teichoic acid in the cell wall. A decrease in teichoic acid synthesis coupled with the rapid turnover of this polymer led to a progressive loss until less than 20% of the level found in wild-type rods remained in spheres. Extracts of cells grown at 45 C contained amounts of the enzymes involved in the biosynthesis and glucosylation of teichoic acids that were equal to or greater than those found in normal rods. Cell walls of the spheres were deficient also in the endogenous autolytic enzyme (N-acyl muramyl-l-alanine amidase). Genetic analysis of the mutant by PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation demonstrated that the lesion responsible for these effects (tag-1) is tightly linked to the genes which regulate the glucosylation of teichoic acid in the mid-portion of the chromosome of B. subtilis.     

Restriction fragments that exert promoter activity during postexponential growth of Bacillus subtilis.

Two restriction fragments of Bacillus subtilis DNA were identified which caused the cat-86 gene present on the promoter cloning plasmid pPL703 to be activated predominantly during postexponential growth of host cells. The postexponential increase was observed in both sporulation-positive strains and in a spoOA mutant of B. subtilis. However, the postexponential increase in the cat-86 gene product, chloramphenicol acetyltransferase, was diminished or not observed when the plasmid-containing cells were grown in the presence of excess glucose. The promoter-containing fragment, designated as 33, was mapped to a site on the B. subtilis chromosome adjacent to hisA. The other fragment, 14, mapped to a site adjacent to ctrA. When present on a high-copy vector, both fragments caused a reduction in the sporulation frequency of host cells. Fragment 33 in high copy number conferred on B. subtilis cells three additional phenotypic changes: brown colony color, intracellular inclusions, and, in a protease-deficient mutant, the production of extracellular protease activity. These activities were observed only in postexponential-phase cultures.     

Genes involved in SkfA killing factor production protect a Bacillus subtilis lipase against proteolysis

Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor.     

Insertion and fate of the cell wall in Bacillus subtilis

Cell wall assembly was studied in autolysin-deficient and -sufficient strains of Bacillus subtilis. Two independent probes, one for peptidoglycan and the other for surface-accessible teichoic acid, were employed to monitor cell surface changes during growth. Cell walls were specifically labeled with N-acetyl-D-[3H]glucosamine, and after growth, autoradiographs were prepared for both cell types. The locations of silver grains revealed that label was progressively lost from numerous sites on the cell cylinders, whereas label was retained on the cell poles, even after several generations. In the autolysin-deficient and chain-forming strain, it was found that the distance between densely labeled poles approximately doubled after each generation of growth. In the autolysin-sufficient strain, it was found that the numbers of labeled cell poles remained nearly constant for several generations, supporting the premise that completed septa and poles are largely conserved during growth. Fluorescein-conjugated concanavalin A was also used to determine the distribution of alpha-D-glucosylated teichoic acid on the surfaces of growing cells. Strains with temperature-sensitive phosphoglucomutase were used because in these mutants, glycosylation of cell wall teichoic acids can be controlled by temperature shifts. When the bacteria were grown at 45 degrees C, which stops the glucosylation of teichoic acid, the cells gradually lost their ability to bind concanavalin A on their cylindrical surfaces, but they retained concanavalin A-reactive sites on their poles. Discrete areas on the cylinder, defined by the binding of fluorescent concanavalin A, were absent when the synthesis of glucosylated teichoic acid was inhibited during growth for several generations at the nonpermissive temperature. When the mutant was shifted from a nonpermissive to a permissive temperature, all areas of the cylinder became able to bind the labeled concanavalin A after about one-half generation. Old cell poles were able to bind the lectin after nearly one generation at the permissive temperature, showing that new wall synthesis does occur in the cell poles, although it occurs slowly. These data, based on both qualitative and quantitative experiments, support a model for cell wall assembly in B. subtilis, in which cylinders elongate by inside-to-outside growth, with degradation of the stress-bearing old wall in wild-type organisms. Loss of wall material, by turnover, from many sites on the cylinder may be necessary for intercalation of new wall and normal length extension. Poles tend to retain their wall components during division and are turned over much more slowly.     

Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.     

Absence of correlation between rates of cell wall turnover and autolysis shown by Bacillus subtilis mutants.

Bacillus subtilis mutants with reduced rates of cell wall autolysis reached a constant rate of wall turnover after a longer lag than the standard strain but eventually showed the same turnover rate. In reverse, a turnover-deficient mutant autolysed at a slightly higher rate than the standard strain. Consequently, there is no correlation between the rates of cell wall turnover and autolysis.     

Protein export during growth of Bacillus subtilis: the effect of extracellular protease deficiency

Exponential phase cultures of a protease-deficient and a protease-proficient strain of Bacillus subtilis , growing in minimal medium with a glucose carbon source, were labelled with ¹⁴C-phenylalanine. Subsequent export of labelled protein was detected in trichloroacetic acid-precipitated supernatant fluid samples. During exponential growth the strains exhibited substantially similar protein export kinetics. However, as confirmed by the presence of a cytoplasmic enzyme in the culture supernatant fluid, protease deficiency was associated with an increased tendency of cells to lyse as they approached stationary phase.     

Structural differentiation of the Bacillus subtilis 168 cell wall.

Exponential-growth-phase cultures of Bacillus subtilis 168 were probed with polycationized ferritin (PCF) or concanavalin A (localized by the addition of horseradish peroxidase conjugated to colloidal gold) to distinguish surface anionic sites and teichoic acid polymers, respectively. Isolated cell walls, lysozyme-digested cell walls, and cell walls treated with mild alkali to remove teichoic acid were also treated with PCF. After labelling, whole cells and walls were processed for electron microscopy by freeze-substitution. Thin sections of untreated cells showed a triphasic, fibrous wall extending more than 30 nm beyond the cytoplasmic membrane. Measurements of wall thickness indicated that the wall was thicker at locations adjacent to septa and at pole-cylinder junctions (P < 0.001). Labelling studies showed that at saturating concentrations the PCF probe labelled the outermost limit of the cell wall, completely surrounding individual cells. However, at limiting PCF concentrations, labelling was observed at only discrete cell surface locations adjacent to or overlying septa and at the junction between pole and cylinder. Labelling was rarely observed along the cell cylinder or directly over the poles. Cells did not label along the cylindrical wall until there was visible evidence of a developing septum. Identical labelling patterns were observed by using concanavalin A-horseradish peroxidase-colloidal gold. Neither probe appeared to penetrate between the fibers of the wall. We suggest that the fibrous appearance of the wall seen in freeze-substituted cells reflects turnover of the wall matrix, that the specificity of labelling to discrete sites on the cell surface is indicative of regions of extreme hydrolytic activity in which alpha-glucose residues of the wall teichoic acids and electronegative sites (contributed by phosphate and carboxyl groups of the teichoic acids and carboxyl groups of the peptidoglycan polymers) are more readily accessible to our probes, and that the wall of exponentially growing B. subtilis cells contains regions of structural differentiation.     

Regulation of bacterial cell walls: correlation between autolytic activity and cell wall turnover in Staphylococcus aureus.

Cell wall turnover was examined in parent and mutant strains of Staphylococcus aureus. Peptidoglycan and teichoic acid were observed to undergo turnover in the wild-type strain during exponential growth; however, the rate of turnover did not decrease when the growth rate slowed, as the culture entered stationary phase. Isolated native cell walls and crude soluble autolytic enzyme were prepared from cells harvested during exponential and postexponential phases of growth. Native cell walls from both phases of growth autolyzed in buffer at identical rates; similarily, crude soluble enzyme from both preparations degraded radioactive cell walls at the same rate. Therefore, the activity of the autolysin in both exponential and postexponential cells was similar. The autolysis of whole cells of a mutant tar-1 was enhanced by 1.0 M NaCl. When 1.0 M NaCl was present under growing conditions, the rate of cell wall turnover was greatly increased. The presence of chloramphenicol, which inhibits whole-cell autolysis, also inhibited turnover. Analysis of the cell wall material recovered from spent medium revealed products consistent with the known mode of action of the endogenous autolysin. It is concluded that cell wall turnover in S. aureus is independent of the stage of culture growth but is dependent instead on the activity of the autolysin.     

Transposon mutagenesis of Pseudomonas aeruginosa exoprotease genes.

Transposon Tn5 was used to generate protease-deficient insertion mutants of Pseudomonas aeruginosa. The presence of Tn5 in the chromosome of P. aeruginosa was demonstrated by transduction and DNA-DNA hybridization. The altered protease production and kanamycin resistance were cotransduced into a wild-type P. aeruginosa strain. A radiolabeled probe of Tn5 DNA hybridized to specific BamHI fragments isolated from the insertion mutants. Two independently isolated Tn5 insertion mutants had reduced protease production, partially impaired elastase activity, and no immunologically reactive alkaline protease.     

Hetero- and autoprocessing of the extracellular metalloprotease (Mpr) in Bacillus subtilis

Most proteases are synthesized as inactive precursors which are processed by proteolytic cleavage into a mature active form, allowing regulation of their proteolytic activity. The activation of the glutamic-acid-specific extracellular metalloprotease (Mpr) of Bacillus subtilis has been examined. Analysis of Mpr processing in defined protease-deficient mutants by activity assay and Western blotting revealed that the extracellular protease Bpr is required for Mpr processing. pro-Mpr remained a precursor form in bpr-deficient strains, and glutamic-acid-specific proteolytic activity conferred by Mpr was not activated in bpr-deficient strains. Further, purified pro-Mpr was processed to an active form by purified Bpr protease in vitro. We conclude that Mpr is activated by Bpr in vivo, and that heteroprocessing, rather than autoprocessing, is the major mechanism of Mpr processing in vivo. Exchange of glutamic acid for serine in the cleavage site of Mpr (S93E) allowed processing of Mpr into its mature form, regardless of the presence of other extracellular proteases, including Bpr. Thus, a single amino acid change is sufficient to convert the Mpr processing mechanism from heteroprocessing to autoprocessing.     

Cellular lysis in Bacillus subtilis; the affect of multiple extracellular protease deficiencies

Cellular lysis properties of strains of Bacillus subtilis deficient in the synthesis of extracellular proteases was investigated. In all cases, extracellular protease deficiency was found to increase the extent of cellular lysis of batch cultured strains following the transition to stationary phase, the time at which extracellular degradative enzymes are secreted in large quantities. The data indicates that the major extracellular proteases, NprE and AprE, are primarily responsible for the control of this autolytic activity in B. subtilis and has implications for the use of extracellular protease-deficient strains as hosts for the production of heterologous proteins.