Metabolic roles of a Rhodobacter sphaeroides member of the sigma32 family

Abstract

We report the role of a gene (rpoH) from the facultative phototroph Rhodobacter sphaeroides that encodes a protein (sigma37) similar to Escherichia coli sigma32 and other members of the heat shock family of eubacterial sigma factors. R. sphaeroides sigma37 controls genes that function during environmental stress, since an R. sphaeroides deltaRpoH mutant is approximately 30-fold more sensitive to the toxic oxyanion tellurite than wild-type cells. However, the deltaRpoH mutant lacks several phenotypes characteristic of E. coli cells lacking sigma32. For example, an R. sphaeroides deltaRpoH mutant is not generally defective in phage morphogenesis, since it plates the lytic virus RS1, as well as its wild-type parent. In characterizing the response of R. sphaeroides to heat, we found that its growth temperature profile is different when cells generate energy by aerobic respiration, anaerobic respiration, or photosynthesis. However, growth of the deltaRpoH mutant is comparable to that of a wild-type strain under each of these conditions. The deltaRpoH mutant mounted a heat shock response when aerobically grown cells were shifted from 30 to 42 degrees C, but it exhibited altered induction kinetics of approximately 120-, 85-, 75-, and 65-kDa proteins. There was also reduced accumulation of several presumed heat shock transcripts (rpoD P(HS), groESL1, etc.) when aerobically grown deltaRpoH cells were placed at 42 degrees C. Under aerobic conditions, it appears that another sigma factor enables the deltaRpoH mutant to mount a heat shock response, since either RNA polymerase preparations from an deltaRpoH mutant, reconstituted Esigma37, or a holoenzyme containing a 38-kDa protein (sigma38) each transcribed E. coli Esigma32-dependent promoters. The lower growth temperature profile of photosynthetic cells is correlated with a difference in heat-inducible gene expression, since neither wild-type cells or the deltaRpoH mutant mount a typical heat shock response after such cultures were shifted from 30 to 37 degrees C.

FIG. 1

Alignment of ς³² family members (generated by using the PILEUP program [5]). Identical amino acids are indicated in boldface. The broken lines above the alignment denote conserved regions of eubacterial sigma factors (20). GenBank accession numbers for ς³² family members are as follows: C. crescentus, U39791; A. tumefaciens, D50828; Z. mobilis, D50832; B. japonicum, U55047; H. influenzae, U32713; P. aeruginosa, D50052; C. freundii, X14960; and E. coli, U00039. RpoH box and helix-turn-helix (H-T-H) sequences are underlined. Gaps introduced to maximize alignment are indicated by dots. Asterisks indicate the end of the protein. Abbreviations: Rsph, R. sphaeroides; Ccre, C. crescentus; Atum, A. tumefaciens; Zmob, Z. mobilis; Bjap, B. japonicum; Hinf, H. influenzae; Paer, P. aeruginosa; Cfre, C. freundii; Ecoh, E. coli.

FIG. 2

Plating efficiency of aerobically grown R. sphaeroides strains at different tellurite concentrations. Viability of wild-type cells and the ΔRpoH mutant is expressed as the number of CFU per milliliter of original culture at each tellurite concentration.

FIG. 3

Protein synthesis after aerobically grown wild-type and ΔRpoH cells are shifted from 30 to 42°C. Strains, temperatures, and sampling times (in minutes [indicated by prime symbols]) after the shift are indicated over the gel. The migration of prestained molecular mass standards (Gibco-BRL) was used to estimate the apparent molecular masses of the indicated proteins.

FIG. 4

Relative synthesis rates of selected proteins before and after aerobically grown cells are shifted to 42°C. The x axes show the minutes after exposure to 42°C (−1 indicates cells sampled 1 min before the temperature shift). The y axes show the relative pixel intensity of individual proteins in Fig. 5. Wild-type (▭) and ΔRpoH () cells are shown.

FIG. 5

Transcript levels before and 30 min after aerobically grown wild-type and ΔRpoH cells were shifted to 42°C. (A) Primer extension analysis (∼8 μg of RNA per lane) of the rrnB, rpoD P_HS, and groESL₁ transcripts at the indicated temperatures. (B) Primer extension analysis (∼8 μg of RNA per lane) of cycA P1-specific transcripts at the indicated temperatures. (C) Transcript abundance (pixel intensity) from panels A and B. Induction ratios denote the increase at 42°C relative to the 30°C level. (D) Comparison of potential R. sphaeroides promoters with the E. coli ς³² consensus sequence; matches are denoted by boldface type.

FIG. 6

Transcription of E. coli heat shock promoters, dnaK P₁, htpG, and rpoD P_HS, by mixtures of R. sphaeroides RNA polymerase holoenzymes. Wild-type (w.t.) and ΔrpoH R. sphaeroides RNA polymerase (RNAP) were used (+). The RNA1 transcript is a ς⁷⁰-dependent product from the origin of DNA replication on all templates (21).

FIG. 7

R. sphaeroides Eς³⁸ recognizes E. coli dnaK P₁. RNA polymerase holoenzymes were reconstituted by adding potential sigma factors (lanes 1 to 28) from the ΔRpoH mutant to a core RNA polymerase preparation from the ΔRpoH mutant (21). The protein in lane 20 was the 38-kDa polypeptide (ς³⁸) that allowed core RNA polymerase to transcribe the cycA P1 heat shock promoter (21).

FIG. 8

Photosynthetic growth of wild-type cells (top) and the ΔRpoH mutant (bottom) at 30°C and after a temperature shift from 30 to 37°C. Photosynthetic growth of wild-type cells (•) and those lacking ς³⁷ (○) at 30°C and the response seen when wild-type cells (+) or the ΔRpoH (✠) mutant are shifted from 30 to 37°C are shown. The broken vertical line indicates when the photosynthetic cultures of wild-type and ΔRpoH cells were placed at 37°C.

FIG. 9

Protein synthesis before and after photosynthetic wild-type and ΔRpoH cells are shifted from 30 to 37°C. Temperatures and sampling times (in minutes [indicated by prime symbols]) after the shift are indicated over the gel. The migration of molecular mass standards (Gibco-BRL) indicated to the left of the gel were used to estimate the apparent sizes of the indicated proteins.