Siger Holsappel

Protein secretion and possible roles for multiple signal peptidases for precursor processing in Bacilli

Bacillus subtilis is one of the best known Gram-positive bacteria at both the genetic and physiological level. The entire sequence of its chromosome is known and efficient tools for the genetic modification of this bacterium are available. Moreover, B. subtilis and related Bacillus species are widely used in biotechnology, in particular for the production of secreted enzymes. Although bacilli can secrete large amounts of several native enzymes, the use of these bacteria for the production of heterologous enzymes has frequently resulted in low yields. Here we describe the identification of several components of the Bacillus protein secretion machinery. These components can now be engineered for optimal protein secretion. Special emphasis is given on type I signal peptidases, which remove signal peptides from secretory precursor proteins. Five genes specifying such enzymes (sip, for signal peptidase) are present on the B. subtilis chromosome. Although none of the sip genes is essential by itself, a specific combination of mutations in these genes is lethal. The expression pattern of some of the sip genes coincides with that of many secretory proteins, which seems to reflect an adaptation to high demands on the secretion machinery. Although the various B. subtilis type I signal peptidases have at least partially overlapping substrate specificities, clear differences in substrate preferences are also evident. These observations have implications for the engineering of the processing apparatus for improved secretion of native and heterologous proteins by Bacillus.

PLASMID DELETION FORMATION BETWEEN SHORT DIRECT REPEATS IN BACILLUS-SUBTILIS IS STIMULATED BY SINGLE-STRANDED ROLLING-CIRCLE REPLICATION INTERMEDIATES

SummaryThe effects of the rolling-circle mode of replication and the generation of single-stranded DNA (ss DNA) on plasmid deletion formation between short direct repeats in Bacillus subtilis were studied. Deletion units consisting of direct repeats (9, 18, or 27 bp) that do or do not flank inverted repeats (300 bp) were introduced into various plasmid replicons that generate different amounts of ss DNA (from 0% to 40% of the total plasmid DNA). With ss DNA-generating rolling-circle-type plasmids, deletion frequencies between the direct repeats were 3- to 13-fold higher than in plasmids not generating ss DNA. When the direct repeats flanked inverted repeats the deletion frequencies in ss DNA-generating plasmids were increased by as much as 20- to 140-fold. These results support models for deletion formation based on template-switching errors during complementary strand synthesis of rolling-circle-type plasmids. The structural instability (deletion formation between short direct repeats) of the ss DNA-generating plasmid pTA1060 in B. subtilis was very low in the presence of a functional initiation site for complementary strand synthesis (minus origin). This observation suggests that it will be possible to develop stable host-vector cloning systems for B. subtilis.

SporeWeb: an interactive journey through the complete sporulation cycle of Bacillus subtilis

Bacterial spores are a continuous problem for both food-based and health-related industries. Decades of scientific research dedicated towards understanding molecular and gene regulatory aspects of sporulation, spore germination and spore properties have resulted in a wealth of data and information. To facilitate obtaining a complete overview as well as new insights concerning this complex and tightly regulated process, we have developed a database-driven knowledge platform called SporeWeb (http://sporeweb.molgenrug.nl) that focuses on gene regulatory networks during sporulation in the Gram-positive bacterium Bacillus subtilis. Dynamic features allow the user to navigate through all stages of sporulation with review-like descriptions, schematic overviews on transcriptional regulation and detailed information on all regulators and the genes under their control. The Web site supports data acquisition on sporulation genes and their expression, regulon network interactions and direct links to other knowledge platforms or relevant literature. The information found on SporeWeb (including figures and tables) can and will be updated as new information becomes available in the literature. In this way, SporeWeb offers a novel, convenient and timely reference, an information source and a data acquisition tool that will aid in the general understanding of the dynamics of the complete sporulation cycle.

Time-resolved dual RNA-seq reveals extensive rewiring of lung epithelial and pneumococcal transcriptomes during early infection

BackgroundStreptococcus pneumoniae, the pneumococcus, is the main etiological agent of pneumonia. Pneumococcal infection is initiated by bacterial adherence to lung epithelial cells. The exact transcriptional changes occurring in both host and microbe during infection are unknown. Here, we developed a time-resolved infection model of human lung alveolar epithelial cells by S. pneumoniae and assess the resulting transcriptome changes in both organisms simultaneously by using dual RNA-seq.ResultsFunctional analysis of the time-resolved dual RNA-seq data identifies several features of pneumococcal infection. For instance, we show that the glutathione-dependent reactive oxygen detoxification pathway in epithelial cells is activated by reactive oxygen species produced by S. pneumoniae. Addition of the antioxidant resveratrol during infection abates this response. At the same time, pneumococci activate the competence regulon during co-incubation with lung epithelial cells. By comparing transcriptional changes between wild-type encapsulated and mutant unencapsulated pneumococci, we demonstrate that adherent pneumococci, but not free-floating bacteria, repress innate immune responses in epithelial cells including expression of the chemokine IL-8 and the production of antimicrobial peptides. We also show that pneumococci activate several sugar transporters in response to adherence to epithelial cells and demonstrate that this activation depends on host-derived mucins.ConclusionsWe provide a dual-transcriptomics overview of early pneumococcal infection in a time-resolved manner, providing new insights into host-microbe interactions. To allow easy access to the data by the community, a web-based platform was developed (http://dualrnaseq.molgenrug.nl). Further database exploration may expand our understanding of epithelial–pneumococcal interaction, leading to novel antimicrobial strategies.

DNA-microarrays and food-biotechnology.

The recent boost in bacterial genome sequences (e.g. Kunst et al. 1997; Bolotin et al. 1999) now enables true ‘functional genomics’ studies, including transcriptome- proteome- and metabolome analyses as well as structural genomics. For these areas novel high-throughput analytical methods have been developed, such as DNA-microarrays and improved 2D-electrophoresis methods combined with MALDITOF analyses. The large number of experimental data generated are gathered into large data-bases, the interpretation of which will greatly depend on novel bioinformatics methodologies, which should enable linking of the databases and facilitate classification and interpretation of the results.

Next-Generation Whole-Genome Sequencing of Eight Strains of Bacillus cereus, Isolated from Food

ABSTRACT Bacillus cereus can contaminate food and cause emetic and diarrheal foodborne illness. Here, we report whole-genome sequences of eight strains of B. cereus, isolated from different food sources.

The 172 kb prkA-addAB region from 83 degrees to 97 degrees of the Bacillus subtilis chromosome contains several dysfunctional genes, the glyB marker, many genes encoding transporter proteins, and the ubiquitous hit gene

A 171812 bp nucleotide sequence between prkA and addAB (83 degrees to 97 degrees) on the genetic map of the Bacillus subtilis 168 chromosome was determined and analysed. An accurate physical/genetic map of this previously poorly described chromosomal region was constructed. One hundred and seventy open reading frames (ORFs) were identified on the DNA fragment. These include the previously described genes cspB, glpPFKD, spoVR, phoAIV, papQ, citRA, sspB, prsA, hpr, pbpF, hemEHY, aprE, comK and addAB. ORF yhaF in this region corresponds to the glyB marker. Among the striking features of this region are: an abundance of genes encoding (putative) transporter proteins, several dysfunctional genes, the ubiquitous hit gene, and five multidrug-resistance-like genes. These analyses have also revealed the existence of numerous paralogues of ORFs in this region: about two-thirds of the putative genes seem to have at least one paralogue in the B. subtilis genome.

A 22 kb DNA sequence in the cspS-glpPFKD region at 75 degrees on the Bacillus subtilis chromosome

A 21808 bp nucleotide sequence at 75 degrees on the genetic map of the Bacillus subtilis chromosome was determined. The sequence of this region is adjacent to the glpPFKD operon involved in glycerol utilization. Twenty-six ORFs were identified, one of which corresponds to the cspB gene, encoding a cold-shock protein. Seventeen of the deduced protein sequences of these ORFs displayed significant homology to known proteins in the data banks. One putative operon was identified, consisting of five ORFs, that is probably involved in the uptake and processing of copper. The location of cspB in this sequence does not confirm the genetic mapping data, indicating that the gene is closely linked to comK, which is located at 80 degrees on the B. subtilis chromosome.

Genome Sequences of 12 Spore-Forming Bacillus Species, Comprising Bacillus coagulans, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus sporothermodurans, and Bacillus vallismortis, Isolated from Foods

ABSTRACT Here, we report the draft genomes of twelve isolates of five different Bacillus species, all spore-forming, Gram-positive bacteria.

Draft Genome Sequences of Seven Thermophilic Spore-Forming Bacteria Isolated from Foods That Produce Highly Heat-Resistant Spores, Comprising Geobacillus spp., Caldibacillus debilis, and Anoxybacillus flavithermus

ABSTRACT Here, we report the draft genomes of five strains of Geobacillus spp., one Caldibacillus debilis strain, and one draft genome of Anoxybacillus flavithermus, all thermophilic spore-forming Gram-positive bacteria.