Article Figures & Data
Figures
- Fig. 1.
Scanning electron micrograph of aerial hyphae of the whiA disruption mutant J2401 (A) and the wild-type strain M145 (B). In contrast to the wild-type chains of unicellular spores, the whiA mutant exhibits slightly coiled “stalks” surmounted by coiled aerial hyphae that are often 100 μm or more long (the coiled segment shown here is ca. 150 μm long) and no indentations that might indicate sporulation septa. In this example, a number of branches (arrows) have emerged from the coiled hypha. We have not observed this previously in whiA mutants studied by phase-contrast microscopy, and we suspect that the branches are the equivalent of germ tubes, which are sometimes seen to emerge from spores in spore chains. Bar, 5 μm.
- Fig. 2.
The whiA region and its comparison with similar regions of other bacterial chromosomes. (A) Subcloning analysis by complementation of strain C13 and schematic representation of the arrangement of the genes. Although one possible start codon for ORF2 overlaps the stop codon for ORF1, the codon usage for the region between this and the next possible start codon 114 bp downstream would not be typical for Streptomyces coding sequences, with a G+C content in the third position of only 73% compared to >90% for most genes (39). The end of ORF2 and the beginning of ORF3 are separated by only 7 bp. There are two likely ATG start codons forwhiA, separated by 3 bp: the more upstream of them is 7 bp from a predicted ribosome-binding site and overlaps by 10 bp the end of ORF3; the other ATG codon (13 bp from the ribosome-binding site) overlaps the stop codon for ORF3. The stop codon of whiA and the putative start codon of the mini-ORF are separated by 11 bp, and the stop codon of the mini-ORF and the start codon for ORF5 are separated by 87 bp. In this noncoding intergenic region, there are some inverted repeats that might act as transcription terminators. (B) Arrangement of related genes from other bacteria. The genes are shaded to indicate familial relationships.
- Fig. 3.
Alignment of WhiA-like proteins from diverse gram-positive bacteria. The sequences compared are fromMycoplasma pneumoniae (mycpne; accession number AAB96238 ),Mycoplasma genitalium (mycgen; accession number AAC71321 ),Mycobacterium tuberculosis (myctbc; accession numberCAB02171 ), S. coelicolor (scwhia; this paper), andBacillus subtilis (bacsub; accession number CAB08059 ). TheM. tuberculosis protein is the most similar to WhiA (ca. 70% amino acid identity); the others, all from low-G+C bacteria, ranged from 20 to 27% identity. The consensus sequence is based on the occurrence of identical residues in at least three of the five sequences (black boxes). Grey boxes indicate residues similar to consensus residues. The positions of point mutations, frameshifts (fs), and the insertion of the hyg cassette in J2401 discussed in the text are marked underneath the consensus sequence. Regions predicted to form α-helices in the C-terminal part of the S. coelicolor WhiA are overlined.
- Fig. 4.
Sequence (nucleotides 2079 to 2343 from AF106003 ) at the overlap of ORF3 and whiA, showing the promoter regions and the transcription start point of whiA (bold), the stop codon of ORF3 (overlined), the putative ribosome-binding site forwhiA (boxed), the two putative ATG start codons forwhiA (underlined), and the proposed translational overlap between ORF3 and whiA (only the translation of the last line of the nucleotide sequence is shown). At the bottom, the sequence corresponding to bp 2291 to 2343 (and its translation) is reproduced, showing the mutation found in mutant C13 (TG→CT shown in bold italics in both sequences). The stop codon of ORF3 in C13 (overlined) is located 11 and 17 bp, respectively, upstream of the two proposed start codons for whiA, whereas in the wild type, ORF3 overlapswhiA. Thus, the mutation in C13 could affect the translational coupling of whiA and ORF3, as well as the efficiency of the ribosome-binding site located upstream of thewhiA start codon.
- Fig. 5.
The developmental up-regulation of thewhiA-specific mRNA is greatly reduced in a whiAmutant. RNA samples were extracted from surface-grown cultures at (left to right) 18, 24, 36, 48, 72, 96, and 120 h (M145) and 24, 36, 48, 72, 96, and 120 h (J2401) and subjected to S1 nuclease protection analysis using radiolabeled probes for the indicated transcripts. The developmental progress of the culture is indicated above the panels (V, vegetative growth; A, aerial mycelium; S, sporulation). Note that the autoradiographs obtained with the whiA probe were exposed for five times as long as the hrdB autoradiographs.
