Characterization of sporulation histidine kinases of Bacillus anthracis

Abstract

The initiation of sporulation in Bacillus species is regulated by the phosphorelay signal transduction pathway, which is activated by several histidine sensor kinases in response to cellular and metabolic signals. Comparison of the protein components of the phosphorelay between Bacillus subtilis and Bacillus anthracis revealed high homology in the phosphorelay orthologs of Spo0F, Spo0B, and Spo0A. The sensor domains of sensor histidine kinases are poorly conserved between species, making ortholog recognition tenuous. Putative sporulation sensor histidine kinases of B. anthracis were identified by homology to the HisKA domain of B. subtilis sporulation sensor histidine kinases, which interacts with Spo0F. Nine possible kinases were uncovered, and their genes were assayed for complementation of kinase mutants of B. subtilis, for ability to drive lacZ expression in B. subtilis and B. anthracis, and for the effect of deletion of each on the sporulation of B. anthracis. Five of the nine sensor histidine kinases were inferred to be capable of inducing sporulation in B. anthracis. Four of the sensor kinases could not be shown to induce sporulation; however, the genes for two of these were frameshifted in all B. anthracis strains and one of these was also frameshifted in the pathogenic pXO1-bearing Bacillus cereus strain G9241. It is proposed that acquisition of plasmid pXO1 and pathogenicity may require a dampening of sporulation regulation by mutational selection of sporulation sensor histidine kinase defects. The sporulation of B. anthracis ex vivo appears to result from any one or a combination of the sporulation sensor histidine kinases remaining.

FIG. 1.

The pathway of phosphoryl transfer in the Bacillus subtilis sporulation phosphorelay.

FIG. 2.

Restriction map of plasmid pORI-Cm. The restriction sites within the multiple cloning site (mcs) that are not unique are indicated by the asterisk. The plasmid confers resistance to 7.5 μg/ml of chloramphenicol to E. coli and B. anthracis and replicates in both hosts via the temperature-sensitive repA replicon of pWV01 (15).

FIG. 3.

Transcription analysis of B. anthracis histidine sensor kinase promoters in B. subtilis. Promoter-lacZ transcriptional fusion constructs were integrated at the B. subtilis amyE locus. Cells were grown in Schaeffer's sporulation medium, and β-galactosidase analyses were carried out at hourly intervals. T0 indicates the time of transition from exponential growth to stationary phase. Symbols: ▵, JH24020 (BA4223); □, JH24014 (BA2291); ▴, JH24019 (BA3702); •, JH24015 (BA1351); ▪, JH24016 (BA1356); ⧫, JH24021 (BA5029); ▾, JH24017 (BA1478); ○, JH24018 (BA2636); ✚, JH24098 (pJM115 vector control). Panels A, B, and C represent high, medium, and low levels of expression, respectively.

FIG. 4.

Transcription analysis of B. anthracis histidine sensor kinase promoters in B. anthracis. Promoter-lacZ transcriptional fusion constructs were carried by the pTCVlac replicative vector (28) in the parental strain 34F2. Cells were grown in LB medium containing kanamycin. Symbols: ▵, JHA1008 (BA4223); □, JHA1010 (BA2291); ▴, JHA1007 (BA3702); •, JHA1011 (BA1351); ▪, JHA1012 (BA1356); ⧫, JHA1009 (BA5029); ▾, JHA1004 (BA1478); ○, JHA1006 (BA2636); ✚, JHA1001 (pTCVlac vector control).). Panels A and B represent high and medium-low levels of expression, respectively.