Nucleotide second messenger-mediated regulation of a muralytic enzyme in Streptomyces.

Peptidoglycan degradative enzymes have important roles at many stages during the bacterial life cycle, and it is critical that these enzymes be stringently regulated to avoid compromising the integrity of the cell wall. How this regulation is exerted is of considerable interest: promoter-based control and protein-protein interactions are known to be employed; however, other regulatory mechanisms are almost certainly involved. In the actinobacteria, a class of muralytic enzymes - the 'resuscitation-promoting factors' (Rpfs) - orchestrates the resuscitation of dormant cells. In this study, we have taken a holistic approach to exploring the mechanisms governing RpfA function using the model bacterium Streptomyces coelicolor and have uncovered unprecedented multilevel regulation that is coordinated by three second messengers. Our studies show that RpfA is subject to transcriptional control by the cyclic AMP receptor protein, riboswitch-mediated transcription attenuation in response to cyclic di-AMP, and growth stage-dependent proteolysis in response to ppGpp accumulation. Furthermore, our results suggest that these control mechanisms are likely applicable to cell wall lytic enzymes in other bacteria.

Resuscitation-promoting factors are cell wall-lytic enzymes with important roles in the germination and growth of Streptomyces coelicolor.

Dormancy is a common strategy adopted by bacterial cells as a means of surviving adverse environmental conditions. For Streptomyces bacteria, this involves developing chains of dormant exospores that extend away from the colony surface. Both spore formation and subsequent spore germination are tightly controlled processes, and while significant progress has been made in understanding the underlying regulatory and enzymatic bases for these, there are still significant gaps in our understanding. One class of proteins with a potential role in spore-associated processes are the so-called resuscitation-promoting factors, or Rpfs, which in other actinobacteria are needed to restore active growth to dormant cell populations. The model species Streptomyces coelicolor encodes five Rpf proteins (RpfA to RfpE), and here we show that these proteins have overlapping functions during growth. Collectively, the S. coelicolor Rpfs promote spore germination and are critical for growth under nutrient-limiting conditions. Previous studies have revealed structural similarities between the Rpf domain and lysozyme, and our in vitro biochemical assays revealed various levels of peptidoglycan cleavage capabilities for each of these five Streptomyces enzymes. Peptidoglycan remodeling by enzymes such as these must be stringently governed so as to retain the structural integrity of the cell wall. Our results suggest that one of the Rpfs, RpfB, is subject to a unique mode of enzymatic autoregulation, mediated by a domain of previously unknown function (DUF348) located within the N terminus of the protein; removal of this domain led to significantly enhanced peptidoglycan cleavage.

Cell wall hydrolases affect germination, vegetative growth, and sporulation in Streptomyces coelicolor.

Peptidoglycan is a major cell wall constituent of gram-positive bacteria. It is a dynamic macromolecule that is actively remodeled to enable cell growth and differentiation through a tightly choreographed interplay of hydrolytic and biosynthetic enzyme activities. The filamentous bacterium Streptomyces coelicolor has a complex life cycle that likely requires considerable cell wall remodeling to enable both extension of vegetative hyphae and formation of differentiated cell types. In silico analysis of the S. coelicolor genome enabled identification of 56 candidate cell wall hydrolase genes. We found that seven of these genes shared a highly conserved 5' untranslated region and were expressed during both vegetative growth and sporulation; four of these genes were selected for more extensive biochemical and biological characterization. The proteins encoded by these genes, termed RpfA, SwlA, SwlB, and SwlC, were confirmed to be hydrolytic enzymes, as they could efficiently cleave S. coelicolor cell walls. Phenotypic analyses revealed that these enzymes are important throughout development; deletion of each hydrolase gene resulted in a mutant strain that was heat sensitive, defective in spore formation, and either altered in vegetative growth or delayed in spore germination. Our results indicate that these enzymes play key roles at multiple stages in the growth and development of S. coelicolor, highlighting both the lack of redundancy in hydrolase activity and the importance of cell wall remodeling in the S. coelicolor life cycle.

Structural transitions induced by the interaction between tRNA(Gly) and the Bacillus subtilis glyQS T box leader RNA.

The T box system regulates expression of amino acid-related genes in Gram-positive bacteria through premature termination of transcription. Synthesis of the full-length mRNA requires stabilization of an antiterminator element in the 5' untranslated leader RNA by the cognate uncharged tRNA. tRNA(Gly)-dependent antitermination of the Bacillus subtilis glyQS gene (encoding glycyl-tRNA synthetase) can be reproduced in a purified in vitro transcription system, indicating that the nascent transcript is sufficient for interaction with the tRNA. Genetic analyses previously demonstrated base pairing of a single codon in the leader RNA with the tRNA anticodon, and between the antiterminator and the tRNA acceptor end. In this study, we established conditions for specific binding of tRNA(Gly) to glyQS leader RNA generated by phage T7 RNA polymerase. Structural mapping studies revealed tRNA(Gly)-induced protection in the glyQS leader RNA at the two known sites of interaction with the tRNA, as well as at other regions between these sites. The proposed tRNA-dependent structural switch between the competing terminator and antiterminator forms of the leader RNA was demonstrated directly. Changes in tRNA(Gly) upon binding to glyQS leader RNA were detected in the anticodon loop, consistent with pairing with the specifier sequence, and in the highly conserved G19 in the D-loop, similar to effects induced by codon-anticodon interaction in the ribosome. This study provides biochemical evidence for direct interaction of tRNA(Gly) with full-length in vitro transcribed glyQS leader RNA, and an initial view of structural modulations of both RNA partners within the complex.

Monitoring uncharged tRNA during transcription of the Bacillus subtilis glyQS gene.

Expression of the Bacillus subtilis glyQS gene, encoding glycyl-tRNA synthetase, depends on stabilization of an antiterminator element during transcription of the 5' region of the mRNA by binding of uncharged tRNA(Gly). The glyQS gene is a member of the T box family of genes, all of which are involved in generation of charged tRNA. Each gene in this family exhibits an increase in readthrough of a termination signal located upstream of the start of the coding sequence in response to a decrease in the ratio of charged to uncharged tRNA. Many structural features of T box RNAs that are necessary for tRNA-dependent antitermination have been defined, but little is known about the timing or sequence of events that lead to a productive interaction with uncharged tRNA and discrimination against charged tRNA. To investigate these issues, transcription complexes were blocked artificially at specific positions along the leader sequence and tested for the ability to recognize tRNA. Although the sequence element that binds the tRNA anticodon is located more than 100 nt before the termination signal, complexes with nascent transcripts extending to just upstream of the termination site were still competent for antitermination. This result indicates that the transcript can fold into a receptive structure in the absence of the tRNA, and that tRNA is not necessary prior to this point. A mimic of charged tRNA(Gly) inhibited antitermination by uncharged tRNA unless the leader RNA-tRNA(Gly) complexes contained the complete antiterminator. These results suggest that the transcription complex can interact with either uncharged or charged tRNA until it approaches the termination point, allowing maximal flexibility in monitoring the ratio of charged to uncharged tRNA.

tRNA requirements for glyQS antitermination: a new twist on tRNA.

Transcription antitermination of the Bacillus subtilis glyQS gene, a member of the T box gene regulation family, can be induced during in vitro transcription in a minimal system using purified B. subtilis RNA polymerase by the addition of unmodified T7 RNA polymerase-transcribed tRNA(Gly). Antitermination was previously shown to depend on base-pairing between the glyQS leader and the tRNA at the anticodon and acceptor ends. In this study, variants of tRNA(Gly) were generated to identify additional tRNA elements required for antitermination activity, and to determine the effect of structural changes in the tRNA. We find that additions to the 3' end of the tRNA blocked antitermination, in agreement with the prediction that uncharged tRNA is the effector in vivo, whereas insertion of 1 nucleotide between the acceptor stem and the 3' UCCA residues had no effect. Disruption of the D-loop/T-loop tertiary interaction inhibited antitermination function, as was previously demonstrated for tRNA(Tyr)-directed antitermination of the B. subtilis tyrS gene in vivo. Insertion of a single base pair in the anticodon stem was tolerated, whereas further insertions abolished antitermination. However, we find that major alterations in the length of the acceptor stem are tolerated, and the insertions exhibited a pattern of periodicity suggesting that there is face-of-the-helix dependence in the positioning of the unpaired UCCA residues at the 3' end of the tRNA for interaction with the antiterminator bulge and antitermination.