Transcriptional regulators of GntR family in Streptomyces coelicolor A3(2): analysis in silico and in vivo of YtrA subfamily.

Transcriptional factors of the GntR family regulate numerous physiological and morphological processes in response to the nutrient state of bacterial cells. The number of GntR transcriptional factors in genomes of soil-dwelling actinomycetes is one of the highest among bacteria, reflecting both the large size of their chromosomes and the complex ecological niche that they occupy. However, very little is known about the roles of GntRs in actinomycete biology. Here, we analyzed the genome of model actinomycete, Streptomyces coelicolor A3(2), in an attempt to gain new insights into the function of GntR family. All 56 GntR proteins of M145 strain were classified into FadR, HutC, MocR, YtrA, and DevA subfamilies according to their secondary structure. We then checked for the presence of GntR orthologs in six other sequenced Streptomyces and one Kitasatospora genomes, revealing that 12 GntRs were conserved in all analyzed strains. Genomic analysis of the less studied YtrA type regulators revealed 160 sequences present in 88 members of Coriobacteridae, Rubrobacteridae, and Actinobacteridae subclasses. These proteins form seven dense clusters on the consensus phylogenetic tree and their genes are usually co-located with the genes for transport proteins. Probable operator sites were identified for orthologous groups of Sco0823 and Sco3812 proteins. All S. coelicolor YtrA-like regulatory genes (SCO0823, SCO1728, SCO3812) were analyzed at transcriptional level, knocked out, and introduced on moderate copy number plasmid in M145 strain. Also, gene SCO0824, a part of putative SCO0823 operon, was studied. Results of these experiments are discussed here.

Generation of a non-sporulating strain of Streptomyces coelicolor A3(2) by the manipulation of a developmentally controlled ftsZ promoter.

The differentiation of Streptomyces aerial hyphae into chains of unigenomic spores occurs through the synchronous formation of multiple FtsZ rings, leading to sporulation septa. We show here that developmental control of ftsZ transcription is required for sporulation in Streptomyces coelicolor A3(2). Three putative ftsZ promoters were detected in the ftsQ-ftsZ intergenic region. In addition, some readthrough from upstream promoter(s) contributed to ftsZ transcription. S1 nuclease protection assays and transcriptional fusions of the ftsZ promoter region to the egfp gene (for green fluorescent protein) provided evidence that ftsZ2p is a developmentally controlled promoter that is specifically upregulated in sporulating aerial hyphae. This upregulation required all the six early regulatory sporulation genes that were tested: whiA, B, G, H, I and J. The DNA sequence of the promoter indicated that it is not part of the developmental regulon that is controlled by the RNA polymerase sigma factor sigma(WhiG). A strain in which the ftsZ2p promoter was inactivated grew normally during vegetative growth and formed aerial mycelium, but was deficient in sporulation septation. Thus, ftsZ2p was dispensable for vegetative growth, but was required for the strain to make sufficient FtsZ to support developmentally controlled multiple cell divisions in aerial hyphae.

Cell division genes ftsQAZ in Escherichia coli require distant cis-acting signals upstream of ddlB for full expression.

A transcriptional reporter fusion has been introduced into the chromosomal ftsZ locus in such a way that all transcription that normally reaches ftsZ can be monitored. The new Phi(ftsZ-lacZ ) fusion yields four times more beta-galactosidase activity than a ddlB-ftsQAZ-lacZ fusion on a lambda prophage vector. A strongly polar ddlB ::Omega insertion prevents contributions from signals upstream of the ftsQAZ promoters and decreases transcription of the chromosomal Phi(ftsZ-lacZ ) fusion by 66%, demonstrating that around two-thirds of total ftsZ transcription require cis-acting elements upstream of ddlB. We suggest that those elements are distant promoters, and thus that the cell division and cell wall synthesis genes in the dcw gene cluster are to a large extent co-transcribed. The ddlB ::Omega insertion is lethal unless additional copies of ftsQA are provided or a compensatory decrease in FtsZ synthesis is made. This shows that ddlB is a dispensable gene, and reinforces the critical role of the FtsA/FtsZ ratio in septation. Using the new reporter fusion, it is demonstrated that ftsZ expression is not autoregulated.

A developmentally regulated gene encoding a repressor-like protein is essential for sporulation in Streptomyces coelicolor A3(2).

whiH is one of several known loci specifically needed for the orderly multiple sporulation septation of aerial hyphae of Streptomyces coelicolor A3(2) and for the expression of at least some late sporulation genes. DNA complementing whiH mutants was located immediately upstream on hrdB, which encodes the principal sigma factor of S. coelicolor. Sequencing revealed a gene whose disruption gave rise to a typical whiH mutant phenotype. Four whiH mutants contained base changes or a frameshift in this gene. The deduced product of whiH related to a large family of bacterial regulatory proteins, the most similar being several repressors (such as GntR of Bacillus subtilis) responsive to carboxylate-containing intermediates in carbon metabolism. Transcription of whiH was initiated at a single promoter, PwhiH. Levels of whiH mRNA were developmentally regulated, increasing sharply when aerial mycelium was present, and reaching a maximum approximately when spores were first detectable. Transcript levels were markedly increased in a whiH mutant, indicating the possible involvement of WhiH in negative regulation of its own production. PwhiH was directly dependent on the sigma factor encoded by another sporulation gene, whiG, as shown by in vivo and in vitro transcription analysis.

Developmental regulation of transcription of whiE, a locus specifying the polyketide spore pigment in Streptomyces coelicolor A3 (2)

whiE is a complex locus that specifies the polyketide spore pigment in Streptomyces coelicolor A3(2). Two divergently oriented promoters, whiEP1 and whiEP2, were identified in the whiE gene cluster, and their activities were analyzed during colony development in wild-type and sporulation-deficient strains. Both promoters were developmentally regulated; whiEP1 and whiEP2 transcripts were detected transiently at approximately the time when sporulation septa were observed in the aerial hyphae, and transcription from both promoters depended on each of the six known "early" whi genes required for sporulation septum formation (whiA, -B, -G, -H, -I, and -J). Mutation of the late sporulation-specific sigma factor gene, sigF, had no effect on the activity of whiEP1 but blocked transcription from whiEP2. However, sigmaF-containing holoenzyme was not sufficient to direct transcription of whiEP2 in vitro. The whiEP2 promoter controls expression of whiE ORFVIII, encoding a putative flavin adenine dinucleotide-dependent hydroxylase that catalyzes a late tailoring step in the spore pigment biosynthetic pathway. Disruption of whiE ORFVIII causes a change in spore color, from grey to greenish (T.-W. Yu and D. A. Hopwood, Microbiology 141:2779-2791, 1995). Consistent with these observations, construction of a sigF null mutant of S. coelicolor M145 caused the same change in spore color, showing that disruption of sigF in S. coelicolor changes the nature of the spore pigment rather than preventing its synthesis altogether.

Contribution of individual promoters in the ddlB-ftsZ region to the transcription of the essential cell-division gene ftsZ in Escherichia coli.

The essential cell-division gene ftsZ is transcribed in Escherichia coli from at least six promoters found within the coding regions of the upstream ddlB, ftsQ, and ftsA genes. The contribution of each one to the final yield of ftsZ transcription has been estimated using transcriptional lacZ fusions. The most proximal promoter, ftsZ2p, contributes less than 5% of the total transcription from the region that reaches ftsZ. The ftsZ4p and ftsZ3p promoters, both located inside ftsA, produce almost 37% of the transcription. An ftsAp promoter within the ftsQ gene yields nearly 12% of total transcription from the region. A large proportion of transcription (approximately 46%) derives from promoters ftsQ2p and ftsQ1p, which are located inside the upstream ddlB gene. Thus, the ftsQAZ genes are to a large extent transcribed as a polycistronic mRNA. However, we find that the ftsZ proximal region is necessary for full expression, which is in agreement with a recent report that mRNA cleavage by RNase E at the end of the ftsA cistron has a significant role in the contol of ftsZ expression.

Isolation of a carbon starvation regulatory mutant in a marine Vibrio strain.

A carbon starvation-responding lac fusion of the marine Vibrio sp. strain S14 was used as a reporter strain in order to identify genes critical in the regulation of the carbon starvation response. Interestingly, sequence data together with an altered phenotype with respect to the accumulation of guanosine 3',5'-bispyrophosphate (ppGpp) imply that one of the genes (csrS) identified by this approach is an Escherichia coli spoT equivalent. Complementary data suggest that the function encoded by the csrS gene is essential for the successful development of starvation and stress resistance.

Glucose upshift of carbon-starved marine Vibrio sp. strain S14 causes amino acid starvation and induction of the stringent response.

The physiological status of carbon-starved cells of the marine Vibrio sp. strain S14 has been investigated by the analysis of their immediate response to carbon and energy sources. During the first minute after glucose addition to 48-h-starved cells, the pools of ATP and GTP increased rapidly, and the [ATP]/[ADP] ratio reached the level typical for growing cells within 4 min. The total rates of RNA and protein synthesis increased initially but were inhibited 4 to 5 min after glucose addition by the induction of the stringent response. A mutation in the relA gene abolished stringent control during the recovery and significantly prolonged the lag phase, before the starved cells regrew, after the addition of a single source of carbon. However, both the wild-type and the relA cells regrew without a significant lag phase when given glucose supplemented with amino acids. On the basis of these results, it is suggested that carbon-starved cells are deficient in amino acid biosynthesis and that ppGpp and the stringent response are involved in overcoming this deficiency, presumably by depressing the synthesis of amino acid biosynthetic enzymes. Furthermore, the data suggest that the starved cells primarily are starved for energy, and evidence is presented that the step-up in the rate of protein synthesis after refeeding is partially dependent on de novo RNA synthesis.

Stringent control during carbon starvation of marine Vibrio sp. strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene.

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14.

How do non-differentiating bacteria adapt to starvation?

Non-differentiating bacteria adapt to starvation induced growth arrest by a complex turn-on/turn-off pattern of protein synthesis. This response shows distinct similarities with those of spore formation in differentiating organisms. A substantial amount of information on the non-growth biology of non-differentiating bacteria can be derived from studies on Vibrio strains. One important result is that carbon rather than nitrogen or phosphorus starvation leads to the development of a starvation and stress resistant cell in these organisms. Hence, we have attempted to characterize the carbon starvation stimulon. By the use of two-dimensional gel electrophoresis of pulse-labelled cells and transposon mutagenesis, using reporter gene constructs, the identity and function of some members of the carbon starvation stimulon have been elucidated. Moreover, regulatory genes of the starvation response have been identified with these techniques. Current studies primarily address the identity and function of these genes. The role of transcript modification and stability for both long term persistence during starvation as well as the efficient recovery of cells which occurs upon nutrient addition is also addressed. It is suggested that an understanding of the functionality of the translational machinery is essential for the understanding of these adaptive pathways. This contribution also discusses the diversity of the differentiation-like response to starvation in different bacteria and whether a general starvation induced programme exists.

Ribosomes exist in large excess over the apparent demand for protein synthesis during carbon starvation in marine Vibrio sp. strain CCUG 15956.

Carbon starvation induces the development of a starvation- and stress-resistant cell state in marine Vibrio sp. strain S14 (CCUG 15956). The starved cells remain highly responsive to nutrients during prolonged starvation and exhibit instantaneous severalfold increases in the rates of protein synthesis and RNA synthesis when substrate is added. In order to elucidate the physiological basis for the survival of cells that are starved for a long time, as well as the capacity of these cells for rapid and efficient recovery, we analyzed the ribosome content of carbon-starved Vibrio sp. strain S14 cells. By using direct chemical measurements of the amounts of ribosomal particles in carbon-starved cultures, we demonstrated that ribosomes were lost relatively slowly (half life, 79 h) and that they existed in large excess over the apparent demand for protein synthesis. After 24 h of starvation the total rate of protein synthesis was 2.3% of the rate during growth, and after 3 days this rate was 0.7% of the rate during growth; the relative amounts of ribosomal particles at these times were 81 and 52%, respectively. The ribosome population consisted of 90% 70S monoribosomes, and no polyribosomes were detected in the starved cells. The 70S monoribosomes were responsible for the bulk of the protein synthesis during carbon starvation; some activity was also detected in the polyribosome size region on sucrose density gradients. We suggest that nongrowing carbon-starved Vibrio sp. strain S14 cells possess an excess protein synthesis capacity, which may be essential for their ability to immediately initiate an upshift program when substrate is added.

Responses to multiple-nutrient starvation in marine Vibrio sp. strain CCUG 15956.

The response of marine Vibrio sp. strain S14 (CCUG 15956) to long-term (48-h) multiple-nutrient starvation (i.e., starvation for glucose, amino acids, ammonium, and phosphate simultaneously) can be described as a three-phase process. The first phase, defined as the stringent control phase, encompasses an accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and decreases in RNA and protein synthesis during the first 40 min. In the second phase, there is a temporary increase in the rates of RNA and protein synthesis between 1 and 3 h paralleling a decrease in the ppGpp pool. The third phase includes gradual decline in macromolecular synthesis after 3 h. Using two-dimensional gel electrophoresis of pulse-labeled proteins, a total of 66 proteins were identified as starvation inducible (Sti), temporally expressed throughout the three phases of starvation. The inhibition of protein synthesis during the first phase of starvation partly disrupted the subsequent temporally ordered synthesis of starvation proteins and prevented the expression of some late starvation proteins. It was also found that the early temporal class of starvation proteins, which included the majority of the Sti proteins, was the most essential for long-term survival. Vibrio sp. strain S14 cultures prestarved (1 h) for glucose, amino acids, ammonium, or phosphate as well as cultures exposed (1 h) to CdCl2 exhibited enhanced survival during the subsequent multiple-nutrient starvation in the presence of chloramphenicol or rifampin, while heat or the addition of cyclic AMP or nalidixic acid prior to starvation had no effect. It was demonstrated that amino acid starvation and CdCl2 exposure, which induced the stringent response, were the most effective in conferring enhanced survival. A few Sti proteins were common to all starvation conditions. In addition, the total number of proteins induced by multiple-nutrient starvation significantly exceeded the sum of those induced by starvation for each of the individual nutrients.