Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500

Protective antigen (PA) of Bacillus anthracis is being considered as a vaccine candidate against anthrax and its production has been explored in several heterologous host systems. Since the expression approaches tested, introduced adverse issues such as inclusion body formation and endotoxin contamination, the production from B. anthracis is presently considered as a preferred method. In this presentation we will report on the effect of protective antigen expression on the metabolism of the producing train B. anthracis, BH500, by comparing it with a control strain carrying an empty plasmid. The two strains were grown in a bioreactor and RNA-seq analysis of the producing and non-producing strain was performed. Several differences were observed, especially significant were the following: the strain expressing rPA showed increased transcription of sigL, the gene encoding RNA polymerase σ54, sigB, the general stress transcription factor gene and its regulators rsbW and rsbV, as well as the global regulatory repressor ctsR. At the same time there were also decreased expression of intracellular heat stress related genes such as groL, groES, hslO, dnaJ, and dnaK and increased expression of extracellular chaperons csaA and prsA2. Additionally, major central metabolism genes belonging to TCA, glycolysis, PPP, and amino acids biosynthesis were up-regulated in the PA-producing strain which was associated with decreased specific growth rates. The information and the observation acquired from this study will be presented together with possible approaches to create a better producing strain

Spatial localization of bacteria controls coagulation of human blood by ‘quorum acting'

Blood coagulation often accompanies bacterial infections and sepsis and is generally accepted as a consequence of immune responses. Though many bacterial species can directly activate individual coagulation factors, they have not been shown to directly initiate the coagulation cascade that precedes clot formation. Here we demonstrated, using microfluidics and surface patterning, that the spatial localization of bacteria substantially affects coagulation of human and mouse blood and plasma. Bacillus cereus and Bacillus anthracis, the anthrax-causing pathogen, directly initiated coagulation of blood in minutes when bacterial cells were clustered. Coagulation of human blood by B. anthracis required secreted zinc metalloprotease InhA1, which activated prothrombin and factor X directly (not via factor XII or tissue factor pathways). We refer to this mechanism as ‘quorum acting’ to distinguish it from quorum sensing—it does not require a change in gene expression, it can be rapid and it can be independent of bacterium-to-bacterium communication

Genome Engineering in Bacillus anthracis Using Cre Recombinase

Genome engineering is a powerful method for the study of bacterial virulence. With the availability of the complete genomic sequence of Bacillus anthracis, it is now possible to inactivate or delete selected genes of interest. However, many current methods for disrupting or deleting more than one gene require use of multiple antibiotic resistance determinants. In this report we used an approach that temporarily inserts an antibiotic resistance marker into a selected region of the genome and subsequently removes it, leaving the target region (a single gene or a larger genomic segment) permanently mutated. For this purpose, a spectinomycin resistance cassette flanked by bacteriophage P1 loxP sites oriented as direct repeats was inserted within a selected gene. After identification of strains having the spectinomycin cassette inserted by a double-crossover event, a thermo-sensitive plasmid expressing Cre recombinase was introduced at the permissive temperature. Cre recombinase action at the loxP sites excised the spectinomycin marker, leaving a single loxP site within the targeted gene or genomic segment. The Cre-expressing plasmid was then removed by growth at the restrictive temperature. The procedure could then be repeated to mutate additional genes. In this way, we sequentially mutated two pairs of genes: pepM and spo0A, and mcrB and mrr. Furthermore, loxP sites introduced at distant genes could be recombined by Cre recombinase to cause deletion of large intervening regions. In this way, we deleted the capBCAD region of the pXO2 plasmid and the entire 30 kb of chromosomal DNA between the mcrB and mrr genes, and in the latter case we found that the 32 intervening open reading frames were not essential to growth

A New Minimal Replicon of Bacillus anthracis Plasmid pXO1 ▿

An 8,883-bp mini-pXO1 plasmid containing a replicon from Bacillus anthracis pXO1 (181.6 kb) was identified by making large deletions in the original plasmid using a newly developed Cre-loxP system. Portions of the truncated mini-pXO1 were cloned into an Escherichia coli vector unable to replicate in B. anthracis. A 5.95-kb region encompassing three putative genes was identified as the minimal pXO1 fragment required for replication of the resulting recombinant shuttle plasmid (named pMR) in B. anthracis. Deletion analysis showed that the only genes essential for replication were the pXO1-14 and pXO1-16 genes, which are transcribed in opposite directions and encode predicted proteins of 66.5 and 67.1 kDa, respectively. The ORF14 protein contains a helix-turn-helix motif, while the ORF16 upstream region contains attributes of a theta-replicating plasmid origin of replication (Ori), namely, an exclusively A+T-containing segment, five 9-bp direct repeats, an inverted repeat, and a σA-dependent promoter for the putative replication initiator Rep protein (ORF16). Spontaneous mutations generated in the ORF14, ORF16, and Ori regions of pMR during PCR amplification produced a temperature-sensitive plasmid that is unable to replicate in B. anthracis at 37°C. The efficacy of transformation of plasmid-free B. anthracis Ames and Sterne strains by the original pMR was ∼103 CFU/μg, while Bacillus cereus strains 569 and ATCC 10987 were transformed with efficiencies of 104 and 102 CFU/μg, respectively. Around 95% of B. anthracis cells retained pMR after one round of sporulation and germination

A Spontaneous Translational Fusion of Bacillus cereus PlcR and PapR Activates Transcription of PlcR-Dependent Genes in Bacillus anthracis via Binding with a Specific Palindromic Sequence

Transformation of Bacillus anthracis with plasmid pUTE29-plcR-papR carrying the native Bacillus cereus plcR-papR gene cluster did not activate expression of B. anthracis hemolysin genes, even though these are expected to be responsive to activation by the global regulator PlcR. To further characterize the action of PlcR, we examined approximately 3,000 B. anthracis transformants containing pUTE29-plcR-papR and found a single hemolytic colony. The hemolytic strain contained a plasmid having a spontaneous plcR-papR intergenic region deletion. Transformation of the resulting plasmid pFP12, encoding a fused PlcR-PapR protein, into the nonhemolytic B. anthracis parental strain produced strong activation of B. anthracis hemolysins, including phosphatidylcholine-specific phospholipase C and sphingomyelinase. The fused PlcR-PapR protein present in a lysate of B. anthracis containing pFP12 bound strongly and specifically to the double-stranded palindrome 5′-TATGCATTATTTCATA-3′ that matches the consensus PlcR-binding site. In contrast, native PlcR protein in a lysate from a B. anthracis strain expressing large amounts of this protein did not demonstrate binding with the palindrome. The results suggest that the activation of PlcR by binding of a PapR pentapeptide as normally occurs in Bacillus thuringiensis and B. cereus can be mimicked by tethering the peptide to PlcR in a translational fusion, thereby obviating the need for PapR secretion, extracellular processing, retrieval into the bacterium, and binding with PlcR

Structural Basis for Latency and Function of Immune Inhibitor A Metallopeptidase, a Modulator of the Bacillus anthracis Secretome

Immune inhibitor A(InhA)-type metallopeptidases are potential virulence factors secreted by members of the Bacillus cereus group. Two paralogs from anthrax-causing Bacillus anthracis (BaInhA1 and BaInhA2) were shown to degrade host tissue proteins with broad substrate specificity. Analysis of their activation mechanism and the crystal structure of a zymogenic BaInhA2 variant revealed a ∼750-residue four-domain structure featuring a pro-peptide, a catalytic domain, a domain reminiscent of viral envelope glycoproteins, and a MAM domain grafted into the latter. This domain, previously found only in eukaryotes, is required for proper protein expression in B. anthracis and evinces certain flexibility. Latency is uniquely modulated by the N-terminal segment of the pro-peptide, which binds the catalytic zinc through its α-amino group and occupies the primed side of the active-site cleft. The present results further our understanding of the modus operandi of an anthrax secretome regulator.This study was supported in part by grants from European, Spanish, and Catalan agencies (FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; BFU2012-32862; BFU2015-64487R; MDM-2014-0435; BIO2013-49320-EXP; and 2014SGR9), and by the intramural program of the National Institute of Allergy and Infectious Diseases, NIH. The Department of Structural Biology of IBMB is a “María de Maeztu” Unit of Excellence of the Spanish Ministry of Economy and Competitiveness. Funding for traveling and synchrotron data collection was provided in part by ESRFPeer Reviewe

Codon-Optimized Fluorescent Proteins Designed for Expression in Low-GC Gram-Positive Bacteria▿

Fluorescent proteins have wide applications in biology. However, not all of these proteins are properly expressed in bacteria, especially if the codon usage and genomic GC content of the host organism are not ideal for high expression. In this study, we analyzed the DNA sequences of multiple fluorescent protein genes with respect to codons and GC content and compared them to a low-GC gram-positive bacterium, Bacillus anthracis. We found high discrepancies for cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and the photoactivatable green fluorescent protein (PAGFP), but not GFP, with regard to GC content and codon usage. Concomitantly, when the proteins were expressed in B. anthracis, CFP- and YFP-derived fluorescence was undetectable microscopically, a phenomenon caused not by lack of gene transcription or degradation of the proteins but by lack of protein expression. To improve expression in bacteria with low genomic GC contents, we synthesized a codon-optimized gfp and constructed optimized photoactivatable pagfp, cfp, and yfp, which were in contrast to nonoptimized genes highly expressed in B. anthracis and in another low-GC gram-positive bacterium, Staphylococcus aureus. Using optimized GFP as a reporter, we were able to monitor the activity of the protective antigen promoter of B. anthracis and confirm its dependence on bicarbonate and regulators present on virulence plasmid pXO1