The PrsA lipoprotein is essential for protein secretion in Bacillus subtilis and sets a limit for high-level secretion

Mutations of the prsA gene of Bacillus subtilis have indicated that the gene is involved in protein secretion and it encodes a novel component of the cellular secretion machinery. We now demonstrate that the gene product is a membrane-associated lipoprotein, presumably bound to the outer face of the cytoplasmic membrane. Experiments to inactivate the prsA gene with insertions indicated that it is indispensable for viability. The cellular level of PrsA protein was shown to be decreased in prsA mutants with decreased level of exoproteins, consistent with an essential function in protein secretion. An increased amount of cellular PrsA protein was introduced by Increasing the copy number of prsA in B. subtilis. This enhanced, from six- to twofold, the secretion of α-amylases and a protease in strains, which expressed high levels of these exoenzymes. This suggests that PrsA protein is the rate-limiting component of the secretion machinery, a finding that is of considerable biotechnological interest.     

Heterologous protein secretion in Lactococcus lactis is enhanced by the Bacillus subtilis chaperone-like protein PrsA

The Bacillus subtilis lipoprotein PrsA enhances the yield of several homologous and heterologous exported proteins in B. subtilis by being involved in the posttranslocational stage of the secretion process. In this work, we have studied the effect of B. subtilis PrsA on the secretion of Bacillus amyloliquefaciens α-amylase (AmyQ), a target protein for PrsA, and Bacillus licheniformis penicillinase (PenP) a nontarget protein for PrsA, in Lactococcus lactis. Two compatible plasmids were constructed and introduced into L. lactis strain NZ9000: one high copy plasmid, expressing the AmyQ gene (amyQ) or the PenP gene (penP), and one low copy plasmid, expressing the PrsA encoding gene (prsA). When amyQ and prsA were simultaneously expressed under the nisin-inducible promoter P _nisA , Western blotting experiments revealed a 15- to 20-fold increase in the total yield of AmyQ and a sixfold increase in secreted AmyQ activity, compared to a control strain lacking prsA. When expressed under the same induction conditions, PrsA had no effect on the secretion or total yield of PenP. These results show that the secretion yield of some heterologous proteins can be significantly increased in L. lactis when coproduced with the B. subtilis PrsA protein.     

tmRNA-dependent trans-translation is required for sporulation in Bacillus subtilis

Spore formation in Bacillus subtilis is significantly impaired by the deletion of the gene for tmRNA ( ssrA ), which facilitates the trans -translation reaction that rescues stalled ribosomes and degrades incompletely synthesized peptides. Microscopic analysis revealed that the sporulation of most Δ ssrA cells is blocked after forespore formation. Expression analysis of lacZ -fused genes directed by several RNA polymerase σ factors showed that the synthesis of active σ^K, encoded by the sigK gene, is predominantly inhibited in Δ ssrA cells. The defect in σ^K synthesis is attributable to a defect in the skin element excision, which generates the sigK gene, caused in turn by reduced expression of SpoIVCA (recombinase) in Δ ssrA cells.     

Quantitation of the capacity of the secretion apparatus and requirement for PrsA in growth and secretion of alpha-amylase in Bacillus subtilis

Regulated expression of AmyQ alpha-amylase of Bacillus amyloliquefaciens was used to examine the capacity of the protein secretion apparatus of B. subtilis. One B. subtilis cell was found to secrete maximally 10 fg of AmyQ per h. The signal peptidase SipT limits the rate of processing of the signal peptide. Another limit is set by PrsA lipoprotein. The wild-type level of PrsA was found to be 2 x 10(4) molecules per cell. Decreasing the cellular level of PrsA did not decrease the capacity of the protein translocation or signal peptide processing steps but dramatically affected secretion in a posttranslocational step. There was a linear correlation between the number of cellular PrsA molecules and the number of secreted AmyQ molecules over a wide range of prsA and amyQ expression levels. Significantly, even when amyQ was expressed at low levels, overproduction of PrsA enhanced its secretion. The finding is consistent with a reversible interaction between PrsA and AmyQ. The high cellular level of PrsA suggests a chaperone-like function. PrsA was also found to be essential for the viability of B. subtilis. Drastic depletion of PrsA resulted in altered cellular morphology and ultimately in cell death.     

Intracellular serine protease 1 of Bacillus subtilis is formed in vivo as an unprocessed, active protease in stationary cells.

Western immunoblots and assays of Bacillus subtilis extracts showed that intracellular serine protease 1 is produced in a form larger than previously reported, appears not to have undergone N-terminal processing, and is active in the presence or absence of calcium. No evidence for an inactive precursor form of the protease was found.     

Peptidoglycan hydrolysis is required for assembly and activity of the transenvelope secretion complex during sporulation in Bacillus subtilis

Sporulating Bacillus subtilis cells assemble a transenvelope secretion complex that connects the mother cell and developing spore. The forespore protein SpoIIQ and the mother‐cell protein SpoIIIAH interact across the double membrane septum and are thought to assemble into a channel that serves as the basement layer of this specialized secretion system. SpoIIQ is absolutely required to recruit SpoIIIAH to the sporulation septum on the mother‐cell side, however the mechanism by which SpoIIQ is localized has been unclear. Here, we show that SpoIIQ localization requires its partner protein SpoIIIAH and degradation of the septal peptidoglycan (PG) by the two cell wall hydrolases SpoIID and SpoIIP. Our data suggest that PG degradation enables a second mother‐cell‐produced protein to interact with SpoIIQ. Cells in which both mother‐cell anchoring mechanisms have been disabled have a synergistic sporulation defect suggesting that both localization factors function in the secretion complex. Finally, we show that septal PG degradation is critical for the assembly of an active complex. Altogether, these results suggest that the specialized secretion system that links the mother cell and forespore has a complexity approaching those found in Gram‐negative bacteria and reveal that the sporulating cell must overcome similar challenges in assembling a transenvelope complex.     

NucA is required for DNA cleavage during transformation of Bacillus subtilis

We have re-examined the roles of nucA and nin, in the transformation of Bacillus subtilis as conflicting accounts have been presented concerning the importance of these genes for transformation. The present report demonstrates that nucA deficiency lowers the rate of DNA transport and that NucA is needed for the double-strand cleavage of transforming DNA, probably acting directly as an endonuclease. A relative paucity of DNA termini, resulting from the absence of this endonuclease activity, most probably accounts for the decreased transport rate. NucA is a bitopic integral membrane protein, with its C-terminus external to the membrane where it is appropriately located to effect the cleavage of bound transforming DNA. We have also investigated the roles of the known competence genes in the DNA processing that accompanies transformation in B. subtilis. The genes that are required for DNA transport (comEA, comEC and comFA) are also required for the degradation of the non-transforming strand that accompanies internalization, but comEC and comFA are not needed for the double-strand cleavage that occurs external to the cell membrane.     

DnaK chaperone machine and trigger factor are only partially required for normal growth of Bacillus subtilis

While dnaK and tig are the essential components for nascent polypeptide folding in E. coli, deletion did not confer synthetic lethality in B. subtilis, suggesting that under normal growth conditions, another system or mechanism with a specific role prevails. Likewise, survival at high temperature suffered dramatically, resulting from deletion of several sets of heat shock genes, thus during sudden stress various heat shock genes act synergistically to protect the proteins.     

Structure-function analysis of PrsA reveals roles for the parvulin-like and flanking N- and C-terminal domains in protein folding and secretion in Bacillus subtilis

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.     

The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa

Several proteases are secreted by Pseudomonas aeruginosa including elastase, an abundantly secreted neutral zinc-metalloprotease. Elastase (encoded by lasB) is first synthesized with a relatively large propeptide (18 kDa) domain. Here, we present evidence that this propeptide functions as an intramolecular chaperone (IMC) essential for proper maturation of elastase into a hydrolytically active enzyme. An altered elastase allele (lasB6) that encoded an elastase precursor with a precise propeptide deletion was expressed in Escherichia coli, and disrupted cells contained only inactive elastase. However, co-expression of an allele (lasB7) expressing the propeptide as an independent, non-covalently linked protein rescued about one-third of the hydrolytic activity when compared with that obtained with wild-type lasB. Thus, the propeptide was essential for elastase activity and so defined elastase as an IMC-containing protease. We examined the possibility that the propeptide of elastase also plays a role in the localization of the mature protein past the outer bacterial membrane. Expression of lasB6 in P. aeruginosa (lasBΔ) in the absence of the propeptide resulted in production of inactive elastase that accumulated within the cell and was not secreted to the culture medium. When lasB7 co-expressed the non-covalently linked propeptide in the same cell with lasB6, efficient secretion was restored and active elastase was then found in the supernatant. Thus, the propeptide was needed for secretion of the mature protein as well as enzymatic activity. This chaperone-like activity of the propeptide appears to involve a direct interaction between the mature and propeptide sequences, and evidence for this was obtained by demonstrating that the non-covalently attached 18 kDa propeptide was co-precipitated with elastase using elastase antibodies. These results are consistent with a hypothesis that the propeptide domain acts as an IMC to control both enzymatic activity and competence for secretion.     

CodY is required for nutritional repression of Bacillus subtilis genetic competence.

The acquisition of genetic competence by Bacillus subtilis is repressed when the growth medium contains Casamino Acids. This repression was shown to be exerted at the level of expression from the promoters of the competence-regulatory genes srfA and comK and was relieved in strains carrying a null mutation in the codY gene. DNase I footprinting experiments showed that purified CodY binds directly to the srfA and comK promoter regions.     

The essential GTPase YqeH is required for proper ribosome assembly in Bacillus subtilis

Recent work with bacteria and eukaryotes has shown that GTPases play important roles in ribosome assembly. Here we show that the essential GTPase YqeH is required for proper 70S ribosome formation and 30S subunit assembly/stability in Bacillus subtilis.     

Penicillin-binding protein-related factor A is required for proper chromosome segregation in Bacillus subtilis

Previous work has shown that the ponA gene, encoding penicillin-binding protein 1 (PBP1), is in a two-gene operon with prfA (PBP-related factor A) (also called recU), which encodes a putative 206-residue basic protein (pI = 10.1) with no significant sequence homology to proteins with known functions. Inactivation of prfA results in cells that grow slower and vary significantly in length relative to wild-type cells. We now show that prfA mutant cells have a defect in chromosome segregation resulting in the production of approximately 0.9 to 3% anucleate cells in prfA cultures grown at 30 or 37 degrees C in rich medium and that the lack of PrfA exacerbates the chromosome segregation defect in smc and spoOJ mutant cells. In addition, overexpression of prfA was found to be toxic for and cause nucleoid condensation in Escherichia coli.     

A Streptococcus mutans superoxide dismutase that is active with either manganese or iron as a cofactor

The superoxide dismutase produced by Streptococcus mutans OMZ176 during aerobic growth in a chemically defined medium (modified FMC) that was treated with Chelex 100 (to lower trace metal contamination) and supplemented with high purity manganese was purified (162-fold) by heat treatment, ammonium sulfate precipitation, and chromatofocusing chromatography. The superoxide dismutase produced during aerobic growth in the same medium, but without manganese and supplemented with high purity iron, was similarly purified (220-fold). The molecular masses of each holoenzyme were approximately 43,000 with a subunit mass of 20,700, indicating that the enzymes were dimers of two equally sized subunits. The superoxide dismutase from manganese-grown cells was a manganese enzyme (MnSOD) containing 1.2 atoms of manganese and 0.25 atoms of iron/subunit. The superoxide dismutase from iron-grown cells was an iron enzyme (FeSOD) containing 0.07 atoms of manganese and 0.78 atoms of iron/subunit. The amino acid compositions of the MnSOD and the FeSOD were virtually identical, and their amino-terminal sequences were identical through the first 22 amino acids. Dialysis of the FeSOD with o-phenanthroline and sodium ascorbate generated aposuperoxide dismutase with 94% loss of activity; subsequent dialysis of apoenzyme with either manganese sulfate or ferrous sulfate reconstituted activity (recoveries of 37 and 30%, respectively). Electrophoretic determination of cytoplasmic radioiron distribution indicated that (during aerobic growth) manganese prevented insertion of iron into superoxide dismutase, although the iron levels of at least two other cytoplasmic fractions were not altered by manganese. Therefore, S. mutans used the same aposuperoxide dismutase to form either FeSOD or MnSOD, depending upon which metal was available in the culture medium. Such "cambialistic" enzymes (those capable of making a cofactor substitution) may represent a previously unrecognized family of superoxide dismutases.     

CheB is required for behavioural responses to negative stimuli during chemotaxis in Bacillus subtilis

The methyl-accepting chemotaxis protein, McpB, is the sole receptor mediating asparagine chemotaxis in Bacillus subtilis. In this study, we show that wild-type B. subtilis cells contain approximately 2,000 copies of McpB per cell, that these receptors are localized polarly, and that titration of only a few receptors is sufficient to generate a detectable behavioural response. In contrast to the wild type, a cheB mutant was incapable of tumbling in response to decreasing concentrations of asparagine, but the cheB mutant was able to accumulate to low concentrations of asparagine in the capillary assay, as observed previously in response to azetidine-2-carboxylate. Furthermore, net demethylation of McpB is logarithmically dependent on asparagine concentration, with half-maximal demethylation of McpB occurring when only 3% of the receptors are titrated. Because the corresponding methanol production is exponentially dependent on attractant concentration, net methylation changes and increased turnover of methyl groups must occur on McpB at high concentrations of asparagine. Together, the data support the hypothesis that methylation changes occur on asparagine-bound McpB to enhance the dynamic range of the receptor complex and to enable the cell to respond to a negative stimulus, such as removal of asparagine.     

Expression of levansucrase-beta-galactosidase hybrids inhibits secretion and is lethal in Bacillus subtilis

The lacZ gene of Escherichia coli was fused to several positions downstream from the 5' end of the Bacillus subtilis sacB gene, which encodes levansucrase (LS), a sucrose-inducible extracellular enzyme. Effects of hybrid protein expression in B. subtilis were studied. Several fusions were tested, and two significantly interfered with growth of cells and with LS secretion when induced with sucrose. Chromosomal amplification of the fusions, leading to strong expression of the hybrid proteins, completely blocked LS secretion and was lethal for B. subtilis when expression was induced.