A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy - PubMed (original) (raw)

Comparative Study

A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy

C Fabret et al. J Bacteriol. 1998 Dec.

Abstract

A two-component signal transduction system encoded by the yycF and yycG genes is part of an operon containing three genes, yycH, yycI, and yycJ, with no known function and a gene, yycK, coding for an HtrA-like protease. This operon was transcribed during growth, and its transcription shut down as the cells approached stationary phase. This decreased transcription was not Spo0A dependent. The HtrA protease gene was separately controlled during sporulation from a sigmaG promoter. Studies using insertional inactivation plasmids revealed that neither yycF nor yycG could be inactivated, whereas the other genes were inactivated without loss of viability. A temperature-sensitive YycF response regulator mutant was isolated and shown to have an H215P mutation in a putative DNA-binding domain which is closely related to the OmpR family of response regulators. At the nonpermissive temperature, cultures of the mutant strain stopped growth within 30 min, and this was followed by a decrease in optical density. Microscopically, many of the cells appeared to retain their structure while being empty of their contents. The essential processes regulated by this two-component system remain unknown. A search of the genome databases revealed YycF, YycG, and YycJ homologues encoded by three linked genes in Streptococcus pyogenes. The high level of identity of these proteins (71% for YycF) suggests that this system may play a similar role in gram-positive pathogens.

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Figures

FIG. 1

Genetic organization at the B. subtilis yycFGHIJK operon. purA codes for adenylosuccinate synthetase and rocR for a positive regulator controlling arginine utilization. The arrows identify the coding region of the genes. The restriction sites (_Sac_I and _Mlu_I) used in some constructions are indicated. DNA inserts cloned into plasmids are represented by solid lines, with the corresponding names. The original vector for each construction is indicated at the extreme right. ▿, position of insertion of a terminatorless Cmr-encoding gene from pJM105B into the cloned DNA. The 5.4-kb fragment shows the extent of the PCRs used to generate the temperature-sensitive mutant.

FIG. 2

Amino acid sequence alignment of B. subtilis YycG with B. subtilis ResE (SwissProt accession no. P35164) and PhoR (SwissProt accession no. P23545). The predicted transmembrane helix domains are underlined. Conserved motifs present in the histidine kinase family, designated H, N, G1, F, and G2, are indicated by lines above the corresponding sequences.

FIG. 3

Expression of the yycFG and yycK promoters. (A) Growth in SM (open symbols) of JH17022 (▵) or JH17023 (□) and β-galactosidase specific activity of the yycF-lacZ promoter fusions (solid symbols). (B) Growth in SM (open symbols) of JH17023 (□), JH17024 (spo0A strain with the yycF-lacZ fusion integrated into the amyE locus) (◊), JH17025 (spo0A mutant with the fusion in the chromosomal yycF locus) (▿), and β-galactosidase specific activity of the promoter fusion (solid symbols). (C) Effect of variations in the level of the YycF and YycG proteins on the growth in SM (open symbols) and expression of the operon (solid symbols) of JH17040 without IPTG (○), with 50 μM IPTG (▵), with 0.5 mM IPTG (X), and with 1 mM IPTG (+). Growth of JH17023 (□) is plotted as a control. (D) Growth in SM (open symbols) and β-galactosidase specific activity (solid symbols) of JH642 with the yycK-lacZ fusion integrated into the amyE locus (pJC18) (□) or into the yycK locus (pJC20) (○).

FIG. 4

Growth characteristics of the temperature-sensitive strain. Growth curves of the Mu8u5u16 (□) and JH17041 (temperature-sensitive) (▵) strains in LB broth at 30°C (open symbols) or at 47°C (solid symbols). Time zero is when the culture was shifted to 47°C from 30°C.

FIG. 5

Cellular effect of the thermosensitive mutation. Strain JH17041 was grown in LB broth at 30°C to an OD525 of 0.35, when half the culture was shifted at 47°C. Phase-contrast images of cells taken 1.5 h after the shift are shown. A, 30°C (OD525 = 1.7); B, 47°C (OD525 = 0.6).

FIG. 6

Sequence alignment of the DNA-binding domains of B. subtilis YycF and E. coli OmpR (SwissProt accession no. P03025). A schematic diagram of the secondary structure of OmpR is shown (15). The dots mark residues corresponding to the OmpR hydrophobic core. The position of thermosensitive mutation is shaded.

FIG. 7

Organization of the _yycFG_-like genes in S. pyogenes M1 GAS (sequenced at Oklahoma University [

http://www.genome.ou.edu/strep.html

]) and amino acid sequence alignment of the predicted products (YycFGJSt) with the homologs in B. subtilis (YycFGJBs). Conserved motifs in the histidine kinase proteins are indicated by lines above the corresponding sequences.

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A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy - PubMed (original) (raw)