An Alternative and Conserved Cell Wall Enzyme That Can Substitute for the Lipid II Synthase MurG.

The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here, we present the discovery of a cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster, including murG, surprisingly produced lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG homologue, which encodes a putative enzyme that shares only around 31% amino acid sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MglA. Orthologues of mglA are present in 38% of all genomes of Kitasatospora and members of the sister genus Streptomyces CRISPR interference experiments showed that K. viridifaciens mglA can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. All together, these results identify MglA as a bona fide lipid II synthase, thus demonstrating plasticity in cell wall synthesis.IMPORTANCE Almost all bacteria are surrounded by a cell wall, which protects cells from environmental harm. Formation of the cell wall requires the precursor molecule lipid II, which in bacteria is universally synthesized by the conserved and essential lipid II synthase MurG. We here exploit the unique ability of an actinobacterial strain capable of growing with or without its cell wall to discover an alternative lipid II synthase, MglA. Although this enzyme bears only weak sequence similarity to MurG, it can functionally replace MurG and can even do so in organisms that naturally have only a canonical MurG. The observation that MglA proteins are found in many actinobacteria highlights the plasticity in cell wall synthesis in these bacteria and demonstrates that important new cell wall biosynthetic enzymes remain to be discovered.

Application of redD, the transcriptional activator gene of the undecylprodigiosin biosynthetic pathway, as a reporter for transcriptional activity in Streptomyces coelicolor A3(2) and Streptomyces lividans.

redD encodes the transcriptional activator of the biosynthetic pathway for undecylprodigiosin, a red-pigmented, mycelium-bound antibiotic made by Streptomyces coelicolor A3(2) and Streptomyces lividans. A promoterless version of redD preceded by the efficiently used tuf1 ribosome binding site was inserted into two different plasmid vectors, providing a convenient reporter of transcriptional activity in both species. One plasmid, plJ2587, replicates autonomously in both Escherichia coli and streptomycetes, while the other, plJ2585, replicates in E. coli and can be transferred to streptomycetes by conjugation or transformation, whereupon it integrates stably at the chromosomal attachment site for the temperate phage phiC31. The utility of the plasmids in detecting not only transcriptional activity, but also its regulation, was confirmed using the rrnAp, ermEp*, and glnRp promoters. The ability to screen visually and spectrophotometrically for red pigmentation should make the vectors particularly attractive for analysing the regulation of gene expression, and for the isolation of mutants, in both S. coelicolor and S. lividans.

The ram-dependence of Streptomyces lividans differentiation is bypassed by copper.

The onset of morphological differentiation in Streptomyces lividans is intrinsically delayed in comparison to Streptomyces coelicolor, but can be advanced by adding extra copper to the medium. Copper-specific chelators block aerial hyphae formation in both strains illustrating the crucial role of copper in morphogenesis. The S. coelicolor ram cluster was isolated as a clone that complements the copper-dependent differentiation of S. lividans. The S. lividans ram cluster was cloned and shown to be 99.6% identical to the S. coelicolor clone. The difference in development between S. lividans and S. coelicolor could neither be related to functional differences between the two ram clusters nor to differences in the transcription level. In both strains the low level of ramAB transcription correlated with aerial mycelium formation and was coupled to the upstream ORF ramS. An increased ramAB expression level in S. lividans by the introduction of an extra copy of ram stimulated the development. In S. lividans disruption of ramABR resulted in the inability to produce aerial hyphae. Conversely, the identical mutant of S. coelicolor retained its developmental capacities, indicating the presence of a ram-independent developmental route that is not present or not activated in S. lividans. Aerial hyphae and spore formation in the S. lividans ramABR mutant was restored when grown near wild-type strains, suggesting that the ram gene products are involved in transport of a factor essential for normal development. In addition, an elevated copper concentration in the medium also relieved the developmental block of these mutants. These findings suggest that higher copper concentrations render this ram-associated factor obsolete.

ssgA is essential for sporulation of Streptomyces coelicolor A3(2) and affects hyphal development by stimulating septum formation.

The role of ssgA in cell division and development of streptomycetes was analyzed. An ssgA null mutant of Streptomyces coelicolor produced aerial hyphae but failed to sporulate, and ssgA can therefore be regarded as a novel whi gene. In addition to the morphological changes, antibiotic production was also disturbed, with strongly reduced actinorhodin production. These defects could be complemented by plasmid-borne ssgA. In the wild-type strain, transcription of ssgA was induced by nutritional shift-down and was shown to be linked to that of the upstream-located gene ssgR, which belongs to the family of iclR-type transcriptional regulator genes. Analysis of mycelium harvested from liquid-grown cultures by transmission electron microscopy showed that septum formation had strongly increased in ssgA-overexpressing strains in comparison to wild-type S. coelicolor and that spore-like compartments were produced at high frequency. Furthermore, the hyphae were significantly wider and contained irregular and often extremely thick septa. These data underline the important role for ssgA in Streptomyces cell division.

The tylosin resistance gene tlrB of Streptomyces fradiae encodes a methyltransferase that targets G748 in 23S rRNA.

tlrB is one of four resistance genes encoded in the operon for biosynthesis of the macrolide tylosin in antibiotic-producing strains of Streptomyces fradiae. Introduction of tlrB into Streptomyces lividans similarly confers tylosin resistance. Biochemical analysis of the rRNA from the two Streptomyces species indicates that in vivo TlrB modifies nucleotide G748 within helix 35 of 23S rRNA. Purified recombinant TlrB retains its activity and specificity in vitro and modifies G748 in 23S rRNA as well as in a 74 nucleotide RNA containing helix 35 and surrounding structures. Modification is dependent on the presence of the methyl group donor, S-adenosyl methionine. Analysis of the 74-mer RNA substrate by biochemical and mass spectrometric methods shows that TlrB adds a single methyl group to the base of G748. Homologues of TlrB in other bacteria have been revealed through database searches, indicating that TlrB is the first member to be described in a new subclass of rRNA methyltransferases that are implicated in macrolide drug resistance.

Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis.

Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.

Effects of increased and deregulated expression of cell division genes on the morphology and on antibiotic production of streptomycetes.

This paper describes the effects of increased expression of the cell division genes ftsZ, ftsQ, and ssgA on the development of both solid- and liquid-grown mycelium of Streptomyces coelicolor and Streptomyces lividans. Over-expression of ftsZ in S. coelicolor M145 inhibited aerial mycelium formation and blocked sporulation. Such deficient sporulation was also observed for the ftsZ mutant. Over-expression of ftsZ also inhibited morphological differentiation in S. lividans 1326, although aerial mycelium formation was less reduced. Furthermore, antibiotic production was increased in both strains, and in particular the otherwise dormant actinorhodin biosynthesis cluster of S. lividans was activated in liquid- and solid-grown cultures. No significant alterations were observed when the gene dosage of ftsQ was increased. Analysis by transmission electron microscopy of an S. coelicolor strain overexpressing ssgA showed that septum formation had strongly increased in comparison to wild-type S. coelicolor, showing that SsgA clearly influences Streptomyces cell division. The morphology of the hyphae was affected such that irregular septa were produced with a significantly wider diameter, thereby forming spore-like compartments. This suggests that ssgA can induce a process similar to submerged sporulation in Streptomyces strains that otherwise fail to do so. A working model is proposed for the regulation of septum formation and of submerged sporulation.

Transcriptional and functional analysis of the gene for factor C, an extracellular signal protein involved in cytodifferentiation of Streptomyces griseus.

Factor C is an extracellular signal protein involved in cellular differentiation in Streptomyces griseus. Nuclease S1 mapping experiments revealed that transcription of the gene takes place from a single promoter in a developmental-stage specific manner. The latter was also confirmed by in vivo promoter probing. The sequence of its promoter suggests that the gene is not transcribed by the major sigma factor. The cloned gene expressed from its own promoter in low- and high-copy-number vectors restored normal sporulation to a bald mutant of Streptomyces griseus. Computer analysis of the amino acid sequence revealed the presence of a transmembrane localization segment with the N-terminus positioned inside the cell. These data fit well into our working model that points at an important role for factor C in the morphogenesis of Streptomyces griseus.

Growth phase-dependent transcription of the Streptomyces ramocissimus tuf1 gene occurs from two promoters.

The str operon of Streptomyces ramocissimus contains the genes for ribosomal proteins S12 (rpsL) and S7 (rpsG) and for the polypeptide chain elongation factors G (EF-G) (fus) and Tu (EF-Tu) (tuf). This kirromycin producer contains three tuf or tuf-like genes; tuf1 encodes the regular EF-Tu and is located immediately downstream of fus. In vivo and in vitro transcription analysis revealed a transcription start site directly upstream of S. ramocissimus tuf1, in addition to the operon promoter rpsLp. Transcription from these promoters appeared to be growth phase dependent, diminishing drastically upon entry into stationary phase and at the onset of production of the EF-Tu-targeted antibiotic kirromycin. In surface-grown cultures, a second round of tuf1 transcription, coinciding with aerial mycelium formation and kirromycin production, was observed. The tuf1-specific promoter (tuf1p) was located in the intercistronic region between fus and tuf1 by high-resolution S1 mapping, in vitro transcription, and in vivo promoter probing. During logarithmic growth, the tuf1p and rpsLp transcripts are present at comparable levels. In contrast to Escherichia coli, which has two almost identical tuf genes, the gram-positive S. ramocissimus contains only tuf1 for its regular EF-Tu. High levels of EF-Tu may therefore be achieved by the compensatory activity of tuf1p.

The malEFG gene cluster of Streptomyces coelicolor A3(2): characterization, disruption and transcriptional analysis.

The malEFG gene cluster of the Gram-positive mycelial actinomycete Streptomyces coelicolor A3(2) was cloned and sequenced. MalEFG show only limited similarity to homologues involved in maltose and maltodextrin transport in other bacteria. Disruption of malE prevented the utilization of maltose as carbon source. Transcription of malE was induced by maltose and repressed by glucose.

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes.

malR of Streptomyces coelicolor A3(2) encodes a homologue of the Lacl/GalR family of repressor proteins, and is divergently transcribed from the malEFG gene cluster, which encodes components of an ATP-dependent transport system that is required for maltose utilization. Transcription of malE was induced by maltose and repressed by glucose. Disruption or deletion of malR resulted in constitutive, glucose-insensitive malE transcription at a level markedly above that observed in the parental malR+ strain, and overproduction of MalR prevented growth on maltose as carbon source. Consequently, MalR plays a crucial role in both substrate induction and glucose repression of maltose utilization. malR is expressed from a single promoter with transcription initiating at the first G of the predicted GTG translation start codon.

A novel plasmid vector that uses the glucose kinase gene (glkA) for the positive selection of stable gene disruptants in Streptomyces.

We describe an Escherichia coli plasmid, pIJ2581, that can be used for the efficient construction of stable gene disruptants and of gene deletions in Streptomyces. Integration of pIJ2581 derivatives carrying chromosomal sequences is achieved by selecting for plasmid-encoded thiostrepton resistance, while plasmid excision is secured by counter-selection of the pIJ2581 glkA gene, which confers sensitivity to 2-deoxyglucose.

The tuf3 gene of Streptomyces coelicolor A3(2) encodes an inessential elongation factor Tu that is apparently subject to positive stringent control.

In Streptomyces coelicolor A3(2), two genes, tuf1 and tuf3, encode the apparent polypeptide chain elongation factors EF-Tu1 and EF-Tu3, respectively. While tuf1 appears to code for the major EF-Tu, the function of tuf3 is unknown. To assess the role of EF-Tu3, tuf3 was subjected to mutational and transcriptional analyses. Replacement of the 5'-half of tuf3 by an antibiotic resistance cassette had no detectable effect on phenotype, indicating that tuf3 is not essential for growth or differentiation. The transcription start site of tuf3 was located approximately 195 nt upstream of the translation start site. The sequence of the tuf3 promoter (Ptuf3) resembles the consensus for the major class of eubacterial promoters, and Ptuf3 was recognized preferentially by an RNA polymerase fraction enriched in sigma hrdB, the principal sigma factor of S. coelicolor. Nuclease S1 mapping failed to reveal tuf3 transcripts during growth of S. coelicolor in liquid culture, consistent with the apparent absence of EF-Tu3 in total protein extracts of the same strain. However, tuf3 transcription was observed in cultures of S. coelicolor M145 shortly after nutritional shiftdown (which resulted in the disappearance of tuf1 transcripts) and after addition of serine hydroxamate, both of which induce the stringent response. Transcription of tuf3 was also observed in transition-phase and stationary-phase cultures of S. coelicolor J1681, a strain deleted for bldA (which specifies a tRNA(Leu) for the rare leucine codon UUA). In all of these examples, transcription of tuf3 followed the production of ppGpp, consistent with the hypothesis that tuf3 is subject to positive stringent control.

Mapping of genes involved in macromolecular synthesis on the chromosome of Streptomyces coelicolor A3(2).

The genes for the beta, beta', and seven sigma factor subunits of RNA polymerase, for elongation factors EF-Tu1 and EF-Tu3, and for six rRNA operons were mapped on the combined genetic and physical map of the Streptomyces coelicolor chromosome. Like the previously mapped tRNA genes, the RNA polymerase and rRNA genes map to scattered positions. The lack of rRNA operons in the immediate vicinity of the origin of replication (oriC) and the absence of tRNA genes in any of the rRNA operons are novel features of the Streptomyces chromosome.

Transcription analysis of the Streptomyces coelicolor A3(2) rrnA operon.

Transcription start sites and processing sites of the Streptomyces coelicolor A3(2) rrnA operon have been investigated by a combination of in vivo and in vitro transcription analyses. The data from these approaches are consistent with the existence of four in vivo transcription sites, corresponding to the promoters P1-P4. The transcription start sites are located at -597, -416, -334 and -254 relative to the start of the 16S rRNA gene. Two putative processing sites were identified, one of which is similar to a sequence reported earlier in S. coelicolor and other eubacteria. The P1 promoter is likely to be recognized by the RNA polymerase holoenzyme containing sigma hrdB, the principal sigma factor in S. coelicolor. P2 also shares homology with the consensus for vegetative promoters, but has a sequence overlapping the consensus -35 region that is also present in the -35 regions of P3 and P4. The -35 sequence common to P2, P3 and P4 is not similar to any other known consensus promoter sequence. In fast-growing mycelium, P2 appears to be the most frequently used promoter. Transcription from all of the rrnA promoters decreased during the transition from exponential to stationary phase, although transcription from P1 and P2 ceased several hours before that from P3 and P4.

Cloning and sequencing of the tuf genes of Streptomyces coelicolor A3(2).

Two tuf genes are present in Streptomyces coelicolor A3(2), which have been cloned and sequenced. These genes show a high degree of nucleotide sequence identity to the tuf1 and tuf3 genes of Streptomyces ramocissimus: the tuf1 genes are 94% identical, the tuf3 genes 87%. S. coelicolor tuf1 encodes a protein of 396 amino acids, while tuf3 encodes a protein of 391 amino acids.

Three tuf-like genes in the kirromycin producer Streptomyces ramocissimus.

We have identified, cloned and sequenced three tuf-like genes from Streptomyces ramocissimus (Sr.), the producer of the antibiotic kirromycin which inhibits protein synthesis by binding the polypeptide chain elongation factor EF-Tu. The tuf-1 gene encodes a protein with 71% amino acid residues identical to the well characterized elongation factor Tu of Escherichia coli (Ec.EF-Tu). The genetic location of tuf-1 downstream of a fus homologue and the in vitro activity of Sr.EF-Tu1 show that tuf-1 encodes a genuine EF-Tu. The putative Sr.EF-Tu2 and Sr.EF-Tu3 proteins are 69% and 63% identical to Ec.EF-Tu. Homologues of tuf-1 and tuf-3 were detected in all five Streptomyces strains investigated, but tuf-2 was found in S. ramocissimus only. The three tuf genes were expressed in E. coli and used to produce polyclonal antibodies. Western blot analysis showed that Sr.EF-Tu1 was present at all times under kirromycin production conditions in submerged and surface-grown cultures of S. ramocissimus and in germinating spores. The expression of tuf-2 and tuf-3 was, however, below the detection level. Surprisingly, Sr.EF-Tu1 was kirromycin sensitive, which excludes the possibility that EF-Tu is involved in the kirromycin resistance of S. ramocissimus.

A comparative study of the ribosomal RNA operons of Streptomyces coelicolor A3(2) and sequence analysis of rrnA.

S. coelicolor A3(2) contains six ribosomal RNA operons. Here we describe the cloning of rrnA, rrnC and rrnE, thereby completing the cloning of all operons. Southern hybridisation of genomic DNA with a heterologous probe from the E.coli rrnB 16S rRNA gene showed differences in hybridisation among the six rRNA operon-containing bands. The nucleotide sequence of the 16S rRNA gene and the upstream region of rrnA was determined and compared with the corresponding sequence of rrnD, showing that the 16S rRNA genes are 99% identical. Substantial differences were found, however, in the upstream regions corresponding to the P1 and P2 promoters of rrnD. Southern analysis showed that some of the other rRNA operons of S.coelicolor A3(2) also differed in this part of the upstream region.