Haike Antelmann

Proteomics of Protein Secretion by Bacillus subtilis: Separating the “Secrets” of the Secretome

SUMMARY Secretory proteins perform a variety of important“ remote-control” functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which ∼90 extracellular proteins were identified. Analysis of these proteins disclosed various“ secrets of the secretome,” such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only∼ 50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.

TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway.

The recent discovery of a ubiquitous translocation pathway, specifically required for proteins with a twin-arginine motif in their signal peptide, has focused interest on its membrane-bound components, one of which is known as TatC. Unlike most organisms of which the genome has been sequenced completely, the Gram-positive eubacterium Bacillus subtilis contains twotatC-like genes denoted tatCd andtatCy. The corresponding TatCd and TatCy proteins have the potential to be involved in the translocation of 27 proteins with putative twin-arginine signal peptides of which ∼6–14 are likely to be secreted into the growth medium. Using a proteomic approach, we show that PhoD of B. subtilis, a phosphodiesterase belonging to a novel protein family of which all known members are synthesized with typical twin-arginine signal peptides, is secreted via the twin-arginine translocation pathway. Strikingly, TatCd is of major importance for the secretion of PhoD, whereas TatCy is not required for this process. Thus, TatC appears to be a specificity determinant for protein secretion via the Tat pathway. Based on our observations, we hypothesize that the TatC-determined pathway specificity is based on specific interactions between TatC-like proteins and other pathway components, such as TatA, of which three paralogues are present inB. subtilis.

Bacillus subtilis functional genomics: genome-wide analysis of the DegS-DegU regulon by transcriptomics and proteomics

Abstract. The DegS-DegU two-component regulatory system of Bacillus subtilis controls various processes that characterize the transition from the exponential to the stationary growth phase, including the induction of extracellular degradative enzymes, expression of late competence genes and down-regulation of the σD regulon. The degU32(Hy) mutation stabilizes the phosphorylated form of DegU (DegU-P), resulting in overproduction of several extracellular degradative enzymes. In this study, the pleiotropic DegS-DegU regulon was characterized by combining proteomic and transcriptomic approaches. A comparative analysis of wild-type B. subtilis and the degU32(Hy) mutant grown in complex medium was performed during the exponential and in the stationary growth phase. Besides genes already known to be under the control of DegU-P, novel putative members of this regulon were identified. Although the degU32(Hy) mutant is assumed to contain high levels of phosphorylated DegU in the exponential as well as in the stationary growth phase, many genes known to be positively regulated by DegU-P did not show enhanced expression in the mutant strain during exponential growth. This is consistent with the fact that most genes belonging to the DegS-DegU regulon are subject to multiple regulation; this is also reflected in the strong stationary-phase induction of these genes in the mutant strain. As expected, during the exponential growth phase, the σD regulon was expressed at significantly lower levels in the degU32(Hy) mutant than in the wild type.

The Tat pathway exports multiple virulence proteins in the plant pathogen Streptomyces scabies

Streptomyces scabies is one of a group of organisms that causes the economically important disease potato scab. Analysis of the S. scabies genome sequence indicates that it is likely to secrete many proteins via the twin arginine protein transport (Tat) pathway, including several proteins whose coding sequences may have been acquired through horizontal gene transfer and share a common ancestor with proteins in other plant pathogens. Inactivation of the S. scabies Tat pathway resulted in pleiotropic phenotypes including slower growth rate and increased permeability of the cell envelope. Comparison of the extracellular proteome of the wild type and ΔtatC strains identified 73 predicted secretory proteins that were present in reduced amounts in the tatC mutant strain, and 47 Tat substrates were verified using a Tat reporter assay. The ΔtatC strain was almost completely avirulent on Arabidopsis seedlings and was delayed in attaching to the root tip relative to the wild‐type strain. Genes encoding 14 candidate Tat substrates were individually inactivated, and seven of these mutants were reduced in virulence compared with the wild‐type strain. We conclude that the Tat pathway secretes multiple proteins that are required for full virulence.

Toward a Complete Proteome of Bacillus subtilis

Bacillus subtilis is widely regarded as a model organism for the functional genome analysis of Gram-positive bacteria. This is based on two factors: first, the genome sequence that predicts about 4100 open reading frames was completed in 1997 (1) and second, B. subtilis strain 168 is highly amenable to genetic manipulation. Thus, systematic programs have been initiated to elucidate the functions of all the genes with previously unknown functions. These projects are based on the systematic construction of a mutant library that is used for the genome-wide functional analysis to expand our knowledge of B. subtilis physiology and regulatory mechanisms. Data resulting from these programs are compiled in specific databases (e.g., SubtiList, Micado, Sub2D, SubScript, and SPID) including databases for the genome sequence, functional analysis, proteomics, transcriptomics, and two-hybrid protein interactions (2).Bacillus subtilis is widely regarded as a model organism for the functional genome analysis of Gram-positive bacteria. This is based on two factors: first, the genome sequence that predicts about 4100 open reading frames was completed in 1997 (1) and second, B. subtilis strain 168 is highly amenable to genetic manipulation. Thus, systematic programs have been initiated to elucidate the functions of all the genes with previously unknown functions. These projects are based on the systematic construction of a mutant library that is used for the genome-wide functional analysis to expand our knowledge of B. subtilis physiology and regulatory mechanisms. Data resulting from these programs are compiled in specific databases (e.g., SubtiList, Micado, Sub2D, SubScript, and SPID) including databases for the genome sequence, functional analysis, proteomics, transcriptomics, and two-hybrid protein interactions (2).

Handbook of Proteomic Methods

Bacillus subtilis is widely regarded as a model organism for the functional genome analysis of Gram-positive bacteria. This is based on two factors: first, the genome sequence that predicts about 4100 open reading frames was completed in 1997 (1) and second, B. subtilis strain 168 is highly amenable to genetic manipulation. Thus, systematic programs have been initiated to elucidate the functions of all the genes with previously unknown functions. These projects are based on the systematic construction of a mutant library that is used for the genome-wide functional analysis to expand our knowledge of B. subtilis physiology and regulatory mechanisms. Data resulting from these programs are compiled in specific databases (e.g., SubtiList, Micado, Sub2D, SubScript, and SPID) including databases for the genome sequence, functional analysis, proteomics, transcriptomics, and two-hybrid protein interactions (2).Bacillus subtilis is widely regarded as a model organism for the functional genome analysis of Gram-positive bacteria. This is based on two factors: first, the genome sequence that predicts about 4100 open reading frames was completed in 1997 (1) and second, B. subtilis strain 168 is highly amenable to genetic manipulation. Thus, systematic programs have been initiated to elucidate the functions of all the genes with previously unknown functions. These projects are based on the systematic construction of a mutant library that is used for the genome-wide functional analysis to expand our knowledge of B. subtilis physiology and regulatory mechanisms. Data resulting from these programs are compiled in specific databases (e.g., SubtiList, Micado, Sub2D, SubScript, and SPID) including databases for the genome sequence, functional analysis, proteomics, transcriptomics, and two-hybrid protein interactions (2).

Towards a complete proteome of Bacillus subtilis : cytosolic, cell wall-associated and extracellular proteins

Bacillus subtilis is widely regarded as a model organism for the functional genome analysis of Gram-positive bacteria. This is based on two factors: first, the genome sequence that predicts about 4100 open reading frames was completed in 1997 (1) and second, B. subtilis strain 168 is highly amenable to genetic manipulation. Thus, systematic programs have been initiated to elucidate the functions of all the genes with previously unknown functions. These projects are based on the systematic construction of a mutant library that is used for the genome-wide functional analysis to expand our knowledge of B. subtilis physiology and regulatory mechanisms. Data resulting from these programs are compiled in specific databases (e.g., SubtiList, Micado, Sub2D, SubScript, and SPID) including databases for the genome sequence, functional analysis, proteomics, transcriptomics, and two-hybrid protein interactions (2).Bacillus subtilis is widely regarded as a model organism for the functional genome analysis of Gram-positive bacteria. This is based on two factors: first, the genome sequence that predicts about 4100 open reading frames was completed in 1997 (1) and second, B. subtilis strain 168 is highly amenable to genetic manipulation. Thus, systematic programs have been initiated to elucidate the functions of all the genes with previously unknown functions. These projects are based on the systematic construction of a mutant library that is used for the genome-wide functional analysis to expand our knowledge of B. subtilis physiology and regulatory mechanisms. Data resulting from these programs are compiled in specific databases (e.g., SubtiList, Micado, Sub2D, SubScript, and SPID) including databases for the genome sequence, functional analysis, proteomics, transcriptomics, and two-hybrid protein interactions (2).