High-Molecular-Weight Complexes of RsbR and Paralogues in the Environmental Signaling Pathway of Bacillus subtilisDelumeau, Olivier; Chen, Chien-Cheng; Murray, James W.; Yudkin, Michael D.; Lewis, Richard J.
doi: 10.1128/JB.00892-06pmid: 16963570
Bacillus subtilis has developed an intricate signal transduction cascade to respond to the imposition of a variety of stresses on the cell. Reversible protein phosphorylation and the formation of alternative protein-protein complexes modulate the activity of B , the RNA polymerase sigma factor subunit responsible for the transcription of the general stress response genes. Some of the regulators of B , such as RsbR and RsbS, are known to associate in a 25S complex, called the stressosome, that can bind RsbT until RsbT phosphorylates target residues in RsbR and RsbS. To date, the RsbR-RsbS complex appears to be the most upstream component of the B regulatory pathway. This large structure is thought to play an important role in sensing and/or integrating signals from different physical stresses. The roles of the paralogues of RsbR that are found in B. subtilis remain unclear. We describe here how the RsbR paralogues copurify with RsbR from B. subtilis cell lysates, and we demonstrate in vitro that the paralogues form large complexes either with RsbS or with a prepurified RsbR-RsbS binary complex. We conclude from these biochemical studies that stressosomes in B. subtilis cells contain minimally RsbS and all of the RsbT-phosphorylatable RsbR paralogues.
Reporter Metabolite Analysis of Transcriptional Profiles of a Staphylococcus aureus Strain with Normal Phenotype and Its Isogenic hemB Mutant Displaying the Small-Colony-Variant PhenotypeSeggewiss, Jochen; Becker, Karsten; Kotte, Oliver; Eisenacher, Martin; Yazdi, Mohammad Reza Khoschkhoi; Fischer, Andreas; McNamara, Peter; Al Laham, Nahed; Proctor, Richard; Peters, Georg; Heinemann, Matthias; von Eiff, Christof
doi: 10.1128/JB.00774-06pmid: 16980462
In this study, full-genome DNA microarrays based on the sequence of Staphylococcus aureus N315 were used to compare the transcriptome of a clinical S. aureus strain with a normal phenotype to that of its isogenic mutant with a stable small-colony-variant (SCV) phenotype ( hemB :: ermB ). In addition to standard statistical analyses, systems biology advances were applied to identify reporter metabolites and to achieve a more detailed survey of genome-wide expression differences between the hemB mutant and its parental strain. Genes of enzymes involved in glycolytic and fermentative pathways were found to be up-regulated in the hemB mutant. Furthermore, our analyses allowed identification of additional differences between the normal-phenotype S. aureus and the SCV, most of which were related to metabolism. Profound differences were identified especially in purine biosynthesis as well as in arginine and proline metabolism. Of particular interest, a hypothetical gene of the Crp/Fnr family (SA2424) that is part of the arginine-deiminase (AD) pathway, whose homologue in Streptococcus suis is assumed to be involved in intracellular persistence, showed significantly increased transcription in the hemB mutant. The hemB mutant potentially uses the up-regulated AD pathway to produce ATP or (through ammonia production) to counteract the acidic environment that prevails intracellularly. Moreover, genes involved in capsular polysaccharide and cell wall synthesis were found to be significantly up-regulated in the hemB mutant and therefore potentially responsible for the changed cell morphology of SCVs. In conclusion, the identified differences may be responsible for the SCV phenotype and its association with chronic and persistent infections.
Interaction between Coat Morphogenetic Proteins SafA and SpoVIDCosta, Teresa; Isidro, Anabela L.; Moran, Charles P., Jr.; Henriques, Adriano O.
doi: 10.1128/JB.00761-06pmid: 16950916
Morphogenetic proteins such as SpoVID and SafA govern assembly of the Bacillus subtilis endospore coat by guiding the various protein structural components to the surface of the developing spore. Previously, a screen for peptides able to interact with SpoVID led to the identification of a PYYH motif present in the C-terminal half of the SafA protein and to the subsequent demonstration that SpoVID and SafA directly interact. spoVID and safA spores show deficiencies in coat assembly and are lysozyme susceptible. Both proteins, orthologs of which are found in all Bacillus species, have LysM domains for peptidoglycan binding and localize to the cortex-coat interface. Here, we show that the interaction between SafA and SpoVID involves the PYYH motif (region B) but also a 13-amino-acid region (region A) just downstream of the N-terminal LysM domain of SafA. We show that deletion of region B does not block the interaction of SafA with SpoVID, nor does it bring about spore susceptibility to lysozyme. Nevertheless, it appears to reduce the interaction and affects the complex. In contrast, lesions in region A impaired the interaction of SafA with SpoVID in vitro and, while not affecting the accumulation of SafA in vivo, interfered with the localization of SafA around the developing spore, causing aberrant assembly of the coat and lysozyme sensitivity. A peptide corresponding to region A interacts with SpoVID, suggesting that residues within this region directly contact SpoVID. Since region A is highly conserved among SafA orthologs, this motif may be an important determinant of coat assembly in the group of Bacillus spore formers.
The Ser/Thr Protein Kinase PknB Is Essential for Sustaining Mycobacterial GrowthFernandez, Pablo; Saint-Joanis, Brigitte; Barilone, Nathalie; Jackson, Mary; Gicquel, Brigitte; Cole, Stewart T.; Alzari, Pedro M.
doi: 10.1128/JB.00963-06pmid: 16980473
The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc 2 155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs.
Biofilm Formation by Streptococcus pneumoniae: Role of Choline, Extracellular DNA, and Capsular Polysaccharide in Microbial AccretionMoscoso, Miriam; Garcia, Ernesto; Lopez, Rubens
doi: 10.1128/JB.00673-06pmid: 16936041
Streptococcus pneumoniae colonizes the human upper respiratory tract, and this asymptomatic colonization is known to precede pneumococcal disease. In this report, chemically defined and semisynthetic media were used to identify the initial steps of biofilm formation by pneumococcus during growth on abiotic surfaces such as polystyrene or glass. Unencapsulated pneumococci adhered to abiotic surfaces and formed a three-dimensional structure about 25 µm deep, as observed by confocal laser scanning microscopy and low-temperature scanning electron microscopy. Choline residues of cell wall teichoic acids were found to play a fundamental role in pneumococcal biofilm development. The role in biofilm formation of choline-binding proteins, which anchor to the teichoic acids of the cell envelope, was determined using unambiguously characterized mutants. The results showed that LytA amidase, LytC lysozyme, LytB glucosaminidase, CbpA adhesin, PcpA putative adhesin, and PspA (pneumococcal surface protein A) mutants had a decreased capacity to form biofilms, whereas no such reduction was observed in Pce phosphocholinesterase or CbpD putative amidase mutants. Moreover, encapsulated, clinical pneumococcal isolates were impaired in their capacity to form biofilms. In addition, a role for extracellular DNA and proteins in the establishment of S. pneumoniae biofilms was demonstrated. Taken together, these observations provide information on conditions that favor the sessile mode of growth by S. pneumoniae . The experimental approach described here should facilitate the study of bacterial genes that are required for biofilm formation. Those results, in turn, may provide insight into strategies to prevent pneumococcal colonization of its human host.
Salmonella enterica Serovar Typhimurium Periplasmic Superoxide Dismutase SodCI Is a Member of the PhoPQ Regulon and Is Induced in MacrophagesGolubeva, Yekaterina A.; Slauch, James M.
doi: 10.1128/JB.00706-06pmid: 16980468
Salmonella enterica serovar Typhimurium replicates within host macrophages during the systemic stage of infection. In the macrophage, the bacteria must survive the respiratory burst that produces superoxide. Serovar Typhimurium strain 14028 produces two periplasmic superoxide dismutases, SodCI and SodCII, but only SodCI contributes to virulence. Although we have shown that this is primarily due to differences in the two proteins, evidence suggests differential regulation of the two genes. Using transcriptional sodCI- and sodCII-lac fusions, we show that sodCII is under the control of the RpoS sigma factor, as was known for the Escherichia coli ortholog, sodC . In contrast, we show that sodCI is transcriptionally controlled by the PhoPQ two-component regulatory system, which regulates an array of virulence genes required for macrophage survival. Introduction of a phoP -null mutation into the sodCI fusion strain resulted in a decrease in transcription and loss of regulation. The sodCI - lac fusion showed high-level expression in a background containing a phoQ constitutive allele. The sodCI gene is induced 15-fold in bacteria recovered from either the tissue culture macrophages or the spleens of infected mice. Induction in macrophages is dependent on PhoP. The sodCII fusion was induced three- to fourfold in macrophages and animals; this induction was unaffected by loss of PhoP. Thus, sodCI , which is horizontally transferred by the Gifsy-2 phage, is regulated by PhoPQ such that it is induced at the appropriate time and place to combat phagocytic superoxide.
Cooperative and Critical Roles for Both G Domains in the GTPase Activity and Cellular Function of Ribosome-Associated Escherichia coli EngABharat, Amrita; Jiang, Mengxi; Sullivan, Susan M.; Maddock, Janine R.; Brown, Eric D.
doi: 10.1128/JB.00959-06pmid: 16963571
To probe the cellular phenotype and biochemical function associated with the G domains of Escherichia coli EngA (YfgK, Der), mutations were created in the phosphate binding loop of each. Neither an S16A nor an S217A variant of G domain 1 or 2, respectively, was able to support growth of an engA conditional null. Polysome profiles of EngA-depleted cells were significantly altered, and His 6 -EngA was found to cofractionate with the 50S ribosomal subunit. The variants were unable to complement the abnormal polysome profile and were furthermore significantly impacted with respect to in vitro GTPase activity. Together, these observations suggest that the G domains have a cooperative function in ribosome stability and/or biogenesis.
Crl Facilitates RNA Polymerase Holoenzyme FormationGaal, Tamas; Mandel, Mark J.; Silhavy, Thomas J.; Gourse, Richard L.
doi: 10.1128/JB.01266-06pmid: 16980472
The Escherichia coli Crl protein has been described as a transcriptional coactivator for the stationary-phase sigma factor S . In a transcription system with highly purified components, we demonstrate that Crl affects transcription not only by the E S RNA polymerase holoenzyme but also by E 70 and E 32 . Crl increased transcription dramatically but only when the concentration was low and when Crl was added to prior to assembly with the core enzyme. Our results suggest that Crl facilitates holoenzyme formation, the first positive regulator identified with this mechanism of action.