Marta Perego

The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation

Bacillus subtilis spo0K mutants are blocked at the first step in sporulation. The spo0K strain was found to contain two mutations: one was linked to the trpS locus, and the other was elsewhere on the chromosome. The mutation linked to trpS was responsible for the sporulation defect (spo‐). The unlinked mutation enhanced this sporulation deficiency but had no phenotype on its own. The spo‐ mutation was located in an operon of five genes highly homologous to the oligopeptide transport (Opp) system of Gram‐negative species. Studies with toxic peptide analogues showed that this operon does indeed encode a peptide‐transport system. However, unlike the Opp system of Salmonella typhimurium, one of the two ATP‐binding proteins, OppF, was not required for peptide transport or for sporulation. The OppA peptide‐binding protein, which is periplasmically located in Gram‐negative species, has a signal sequence characteristic of lipo‐proteins with an amino‐terminal lipo‐amino acid anchor. Cellular location studies revealed that OppA was associated with the cell during exponential growth, but was released into the medium in stationary phase. A major role of the Opp system in Gram‐negative bacteria is the recycling of cell‐wall peptides as they are released from the growing peptidoglycan. We postulate that the accumulation of such peptides may play a signaling role in the initiation of sporulation, and that the sporulation defect in opp mutants results from an inability to transport these peptides.

Multiple histidine kinases regulate entry into stationary phase and sporulation in Bacillus subtilis

Protein homology studies identified five kinases potentially capable of phosphorylating the Spo0F response regulator and initiating sporulation in Bacillus subtilis. Two of these kinases, KinA and KinB, were known from previous studies to be responsible for sporulation in laboratory media. In vivo studies of the activity of four of the kinases, KinA, KinC, KinD (ykvD) and KinE (ykrQ), using abrB transcription as an indicator of Spo0A∼P level, revealed that KinC and KinD were responsible for Spo0A∼P production during the exponential phase of growth in the absence of KinA and KinB. In vitro, all four kinases dephosphorylated Spo0F∼P with the production of ATP at approximately the same rate, indicating that they possess approximately equal affinity for Spo0F. All the kinases were expressed during growth and early stationary phase, suggesting that the differential activity observed in growth and sporulation results from differential activation by signal ligands unique to each kinase.

Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis

Sporulation begins coincidentally with the expression of several stationary‐phase‐associated gene products during the transition state of a culture from exponential to stationary phase. Mutations in the stage 0 sporulation genes prevent the expression of these gene products in addition to blocking sporulation. Suppressor mutations in the abrB gene, in a spo0 background, restore stationary‐phase‐associated gene expression but not sporulation. The nature of the abrB gene product was investigated by isolating and sequencing the abrB gene. The abrB gene coded for a 96‐amino‐acid protein (molecular weight 10773) and contained a helix‐turn‐helix structure common to DNA binding proteins. Analysis of expression of the abrB gene using lacZ transcription fusions and direct measurement of mRNA content by hybridization showed that the spo0A gene repressed transcription of the abrB gene. Primer extension analysis of abrB gene mRNA revealed two initiation sites. The downstream site was dramatically repressed in spo0A+ strains, while the upstream site appeared not to be regulated by spo0A. Five abrB mutant alleles were cloned and sequenced. One mutation, abrB 4, resided within the structural gene and continued to overexpress abrB messenger RNA from both promoters. A promoter mutation, abrB 15, reduced transcription from the downstream promoter but not the upstream promoter. Thus, the phenotype of abrB mutations results from inactivation of the abrB gene product or by prevention of its overexpression. The results suggest that the abrB gene codes for a regulator which controls several genes whose products are normally produced during the transition phase between active growth and sporulation. The level of this regulator is, in turn, controlled by the spo0A gene. The pleiotropic phenotypes of spo0A mutants result from uncontrolled overexpression of the abrB regulator.

Multiple protein-aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in B. subtilis.

The initiation of sporulation in B. subtilis is regulated by the Spo0A transcription factor, which is activated by phosphorylation to control developmental switching from the vegetative to the sporulation state. The level of phosphorylation of Spo0A is regulated by the phosphorelay, a signal transduction system based on the protein-histidine kinase-response regulator two-component paradigm. To initiate sporulation, the cell must recognize and interpret a large variety of environmental, metabolic, and cell cycle signals that influence the phosphorylation level of Spo0A. We describe here a family of protein-aspartate phosphatases with activity on Spo0F approximately P, a response regulator component of the phosphorelay, that provide a mechanism for signal recognition and interpretation. These phosphatases function to drain the phosphorelay, lower Spo0A approximately P levels, and prevent sporulation. The integration of diverse environmental signals that affect the initiation of sporulation likely occurs through the competition between opposing activities of protein kinases and protein phosphatases.

Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis

The chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilo‐bases in between have been completely sequenced and functionally characterized. The srfA gene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. The srfA amino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene in srfA encodes a polypeptide homologous to grsT. Four genes are positioned between srfA and sfp, the disruption of which does not affect surfactin biosynthesis.

The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein.

The product of the abrB gene of Bacillus subtilis is an ambiactive repressor and activator of the transcription of genes expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation. Purified AbrB protein binds specifically in a highly co‐operative fashion to fragments of DNA containing the promoters it affects. DNase I footprints of the binding regions in these promoters revealed large protected areas of 50‐120 nucleotides or more depending on the promoter. Methylation protection experiments gave protected guanine residues on only one face of the DNA helix. A consensus sequence could be deduced around these guanine residues that was not found around non‐protected guanine residues in the footprint region. The results suggested that stationary phase functions and sporulation are repressed during active growth by AbrB and other transition state regulators by binding to the affected promoters in a concentration‐dependent manner.

The Enterococcus faecalis fsr Two-Component System Controls Biofilm Development through Production of Gelatinase

Bacterial growth as a biofilm on solid surfaces is strongly associated with the development of human infections. Biofilms on native heart valves (infective endocarditis) is a life-threatening disease as a consequence of bacterial resistance to antimicrobials in such a state. Enterococci have emerged as a cause of endocarditis and nosocomial infections despite being normal commensals of the gastrointestinal and female genital tracts. We examined the role of two-component signal transduction systems in biofilm formation by the Enterococcus faecalis V583 clinical isolate and identified the fsr regulatory locus as the sole two-component system affecting this unique mode of bacterial growth. Insertion mutations in the fsr operon affected biofilm formation on two distinct abiotic surfaces. Inactivation of the fsr-controlled gene gelE encoding the zinc-metalloprotease gelatinase was found to prevent biofilm formation, suggesting that this enzyme may present a unique target for therapeutic intervention in enterococcal endocarditis.

Differential Processing of Propeptide Inhibitors of Rap Phosphatases in Bacillus subtilis

In the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis, the opposing activities of histidine kinases and aspartyl phosphate phosphatases determine the cells decision whether to continue with vegetative growth or to initiate the differentiation process. Regulated dephosphorylation of the Spo0A and Spo0F response regulators allows a variety of negative signals from physiological processes that are antithetical to sporulation to impact on the activation level of the phosphorelay. Spo0F approximately P is the known target of two related phosphatases, RapA and RapB. In addition to RapA and RapB, a third member of the Rap family of phosphatases, RapE, specifically dephosphorylated the Spo0F approximately P intermediate in response to competence development. RapE phosphatase activity was found to be controlled by a pentapeptide (SRNVT) generated from within the carboxy-terminal domain of the phrE gene product. A synthetic PhrE pentapeptide could (i) complement the sporulation deficiency caused by deregulated RapE activity of a phrE mutant and (ii) inhibit RapE-dependent dephosphorylation of Spo0F approximately P in in vitro experiments. The PhrE pentapeptide did not inhibit the phosphatase activity of RapA and RapB. These results confirm previous conclusions that the specificity for recognition of the target phosphatase is contained within the amino acid sequence of the pentapeptide inhibitor.

Aspartyl-phosphate phosphatases deactivate the response regulator components of the sporulation signal transduction system in Bacillus subtilis

Bacteria use two‐component signal transduction systems to sense and respond to their environment. A sensor kinase and a response‐regulator transcription factor work in concert by phosphorylation/dephosphorylation through kinase and phosphatase activities to maintain a level of phosphorylated response regulator commensurate with the level of signal input. Signal input can be accommodated through stimulation of the kinase activity or the phosphatase activity of the two‐component system. With some notable exceptions, the sensor kinases recognize a single stimulatory ligand. A new dimension in the regulation of two‐component signal transduction systems was discovered in the Rap phosphatases which dephosphorylate the Spo0F response‐regulator of Bacillus subtilis independent of the sensor kinases. This family of phosphatases is encoded by at least six chromosomal genes. Although not all of the phosphatases of the family have activity on phosphorylated Spo0F, the two best‐characterized members, RapA and RapB, prevent sporulation by dephosphorylating this response regulator component of the phosphorelay. Phosphatase activity of RapA is regulated by a gene, phrA, in the same transcriptional unit, that encodes a peptide secreted from the cell which may serve as a quorum sensor. Most of the Rap phosphatase operons have a gene coding for a protein with some similarity to PhrA in their transcription units, but it is uncertain whether all of these play a role in regulation. The Rap phosphatases are postulated to be a mechanism for allowing signals other than those that affect the sensor kinases to regulate the signal transduction pathway. They may have been recruited to help regulate sporulation because the multiple signals regulating this process may out‐strip the recognition capacity of the kinases.

Pentapeptide regulation of aspartyl-phosphate phosphatases.

Aspartyl-phosphate phosphatases are integral components of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis. The Rap and Spo0E families of protein phosphatases specifically dephosphorylate the sporulation response regulators Spo0F and Spo0A, respectively. The phosphatases interpret regulatory signals antithetical to sporulation and the Rap phosphatases are subject to inactivation by specific pentapeptides generated from an inactive peptide precursor. Additional regulatory signals are brought about by the complex activation circuit that generates the Phr pentapeptide inhibitors of Rap phosphatases. Phr peptides recognition of the Rap phosphatase targets is remarkably specific. Specificity is dictated by the amino acid sequence of the pentapeptide. The identification of tetratricopeptide repeats in the Rap proteins may explain the mechanism by which Phr peptides bind to and inhibit the activity of Rap phosphatases.