Identification and transcriptional control of Caulobacter crescentus genes encoding proteins containing a cold shock domain

Abstract

The cold shock proteins are small peptides that share a conserved domain, called the cold shock domain (CSD), that is important for nucleic acid binding. The Caulobacter crescentus genome has four csp genes that encode proteins containing CSDs. Three of these (cspA, cspB, and cspC) encode peptides of about 7 kDa and are very similar to the cold shock proteins of other bacteria. Analysis by reverse transcription-PCR of the fourth gene (cspD), which was previously annotated as encoding a 7-kDa protein, revealed that the mRNA is larger and probably encodes a putative 21-kDa protein, containing two CSDs. A search in protein sequences databases revealed that this new domain arrangement has thus far only been found among deduced peptides of alpha-proteobacteria. Expression of each Caulobacter csp gene was studied both in response to cold shock and to growth phase, and we have found that only cspA and cspB are induced by cold shock, whereas cspC and cspD are induced at stationary phase, with different induction rates. The transcription start sites were determined for each gene, and a deletion mapping of the cspD promoter region defined a sequence required for maximal levels of expression, indicating that regulation of this gene occurs at the transcriptional level. Deletion of cspA, but not cspD, caused a reduction in viability when cells were incubated at 10 degrees C for prolonged times, suggesting that cspA is important for adaptation to a low temperature.

FIG. 1.

(A) Amino acid sequence comparison of the three C. crescentus genes containing one CSD with E. coli cspA (GenBank no. AAB18533). (B) Amino acid sequence comparison of the proposed new annotation of cspD from C. crescentus (Cc) with other predicted α-Proteobacteria peptides showing two CSDs. Above the sequences are indicated the two RNA-binding motifs (RNP1 and RNP2). Residues shaded in black indicate conserved residues present in at least 90% of the proteins; those shaded in dark gray are present in at least 60% of the proteins. The GenBank no. and abbreviations are as follows: Agrobacterium tumefaciens (At; AAK87573), Sinorhizobium meliloti (Sm; CAC46297), Brucella melitensis (Bm; AAL51912), Mesorhizobium loti (Ml; BAB47810), Rhodopseudomonas palustris (Rp; ZP_00012063), Magnetospirillum magnetotacticum (Mm; ZP_00049605), Rhodobacter sphaeroides (Rs; ZP_00006193), Novosphingobium aromaticivorans (Na; ZP_00093526), and Rhodospirillum rubrum (Rr; ZP_00013462).

FIG. 2.

Determination of the existence of a longer cspD transcript. (A) The scheme indicates the region of the cspD gene, showing the new proposed coding region (dotted lines), and the originally annotated coding region (dark box). Below is shown the sequence of the proposed cspD coding region, indicating the position of the two primers used in the RT-PCR (arrows). The new (ATG) and annotated (GTG) start sites are boxed. Ribosomal binding site is double underlined. (B) RNA was isolated from mid-log phase cells (lanes 1 and 2) and from cells at 24 h after entry into stationary phase (lanes 3 and 4) and treated with DNase I previous to the experiment. RT-PCR was performed with a pair of oligonucleotides—one that hybridizes close to the ATG of the proposed longer cspD ORF and one at the beginning of the annotated cspD coding region. Control reactions, carried out with Taq DNA polymerase but without reverse transcriptase, yielded no amplified bands (lanes 1 and 3), confirming that there is no contamination of DNA in the samples. The expected 405-nt fragment obtained for both samples is indicated by an arrow.

FIG.3.

(A) Schematic representation of the csp genes. The DNA fragments cloned to the pRKlacZ290 vector in the transcriptional fusions are indicated by bars. The small arrows indicate the primers used to amplify each region from the genome. Some restriction sites are indicated, and the sites inserted by PCR are labeled with an asterisk. (B) Analysis of the cold induction of the csp genes. Cells harboring the transcription fusions of each gene were grown at 30°C up to mid-log phase and were then transferred to different temperatures. Expression of each construct was measured by β-galactosidase activity assays (29) at sequential time points, and the results are shown as relative measurements of induction.

FIG. 4.

Growth-phase-dependent expression of the csp genes. Expression of the csp genes was determined from cells harboring the respective promoter fusions at mid-log phase and at 2 and 24 h after entry into stationary phase. The β-galactosidase activity is expressed in Miller units (29), and growth was monitored by measuring the optical density (OD) at 600 nm.

FIG. 5.

Determination of the transcription start sites of csp genes. Primer extension analysis was carried out with total RNA from exponential-phase cells (L) or stationary-phase cells (S) at 30°C or incubated at 10°C for 1 h (C₁) or 2 h (C₂). The primers were end labeled with ³²P and extended with reverse transcriptase to determine the transcription start sites and were also used in DNA sequencing reactions (shown on the left). Below each panel is shown the respective regulatory regions: cspA (A), cspB (B), cspC (C), and cspD (D). Black arrowheads indicate the transcription start sites, and a white arrowhead indicates the stationary-phase start site of cspD. The most upstream start sites were arbitrarily chosen as position +1, and the −35/−10 sequences are overlined (for clarity, only the most upstream promoters are indicated). The start codons are underlined, and the ribosome-binding sites are double underlined. In panel D, the KpnI restriction site used for the transcription fusion pEL5 (Fig. 6) is boxed, and the position of the BamHI restriction site introduced by PCR in construct pEL4 is shown in parentheses. In panels C and D, the arrows indicate two imperfect direct repeats, and the shaded nucleotides indicate the regulatory sequence of cspD that is also found in cspC.

FIG. 6.

Deletion mapping of cspD promoter region. The scheme of the cspD locus is shown above, indicating the previously annotated coding region (solid line), and the proposed extended coding region (dashed line). The bent arrow indicates the transcription start sites determined by primer extension. Restriction sites are indicated as follows: B, BamHI; K, KpnI; No, NotI; P, PstI. The sites in parentheses were introduced by PCR and are not in the original sequence. The plasmids carrying the constructs were introduced into C. crescentus NA1000, and promoter activity was measured by β-galactosidase assays both in exponential-phase (log) and stationary-phase cells (stat). The results are in Miller units (29) and are the average of at least three independent assays, with the respective standard deviation.

FIG. 7.

Viability of the mutant strains at 10°C. Cells of the parental strain NA1000 (○), NA1000 (ΔcspA) (•), and NA1000 (ΔcspD) (▴) were grown at 30°C up to early log phase and then transferred to 10°C. Aliquots were taken before (0 h) and 24 or 48 h after incubation at 10°C, and serial dilutions were plated to determine the number of CFU. Survival rates were determined relative to NA1000 at each time point.