Persistence of the Chromosome End Regions at Low Copy Number in Mutant Strains of Streptomyces rimosus and Streptomyces lividans

Streptomycetes are important antibiotic producing bacteria that often exhibit genetic instability. One or both ends of the linear Streptomyces chromosome are lost spontaneously, resulting in viable mutant strains sometimes lacking hundreds of genes. We examined some strains of Streptomyces rimosus and Streptomyces lividans, which had been classified as »deletion mutants« and appeared to have lost chromosome end sequences. We discovered that the »deleted« sequences were still present in vegetative mycelium at a very low copy number so that they were normally not detected. The copy number in S. rimosus was estimated as 0.1–1.0 10–3/chromosome. Streptomyces spores contain the disappearing chromosome end sequences at a higher copy number than the vegetative mycelium, promoting their inheritance via spore preparations. This, in effect, represents a separation between germ line and deleted vegetative genomes, which has not been recognised before in Streptomyces, and has practical implications both for strain preservation and genetic studies

Persistence of the Chromosome End Regions at Low Copy Number in Mutant Strains of Streptomyces rimosus and Streptomyces lividans

Streptomycetes are important antibiotic producing bacteria that often exhibit genetic instability. One or both ends of the linear Streptomyces chromosome are lost spontaneously, resulting in viable mutant strains sometimes lacking hundreds of genes. We examined some strains of Streptomyces rimosus and Streptomyces lividans, which had been classified as »deletion mutants« and appeared to have lost chromosome end sequences. We discovered that the »deleted« sequences were still present in vegetative mycelium at a very low copy number so that they were normally not detected. The copy number in S. rimosus was estimated as 0.1–1.0 10–3/chromosome. Streptomyces spores contain the disappearing chromosome end sequences at a higher copy number than the vegetative mycelium, promoting their inheritance via spore preparations. This, in effect, represents a separation between germ line and deleted vegetative genomes, which has not been recognised before in Streptomyces, and has practical implications both for strain preservation and genetic studies

The Genome of Streptococcus mitis B6 - What Is a Commensal?

Streptococcus mitis is the closest relative of the major human pathogen S. pneumoniae. The 2,15 Mb sequence of the Streptococcus mitis B6 chromosome, an unusually high-level beta-lactam resistant and multiple antibiotic resistant strain, has now been determined to encode 2100 genes. The accessory genome is estimated to represent over 40%, including 75 mostly novel transposases and IS, the prophage φB6 and another seven phage related regions. Tetracycline resistance mediated by Tn5801, and an unusual and large gene cluster containing three aminoglycoside resistance determinants have not been described in other Streptococcus spp. Comparative genomic analyses including hybridization experiments on a S. mitis B6 specific microarray reveal that individual S. mitis strains are almost as distantly related to the B6 strain as S. pneumoniae. Both species share a core of over 900 genes. Most proteins described as pneumococcal virulence factors are present in S. mitis B6, but the three choline binding proteins PcpA, PspA and PspC, and three gene clusters containing the hyaluronidase gene, ply and lytA, and the capsular genes are absent in S. mitis B6 and other S. mitis as well and confirm their importance for the pathogenetic potential of S. pneumoniae. Despite the close relatedness between the two species, the S. mitis B6 genome reveals a striking X-alignment when compared with S. pneumoniae

An Important Site in PBP2x of Penicillin-Resistant Clinical Isolates of Streptococcus pneumoniae: Mutational Analysis of Thr338▿

Penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae represents a primary resistance determinant for beta-lactams, and low-affinity PBP2x variants can easily be selected with cefotaxime. Penicillin-resistant clinical isolates of S. pneumoniae frequently contain in their mosaic PBP2x the mutation T338A adjacent to the active site S337, and T338P as well as T338G substitutions are also known. Site-directed mutagenesis has now documented that a single point mutation at position T338 confers selectable levels of beta-lactam resistance preferentially to oxacillin. Despite the moderate impact on beta-lactam susceptibility, the function of the PBP2x mutants appears to be impaired, as can be documented in the absence of a functional CiaRH regulatory system, resulting in growth defects and morphological changes. The combination of low-affinity PBP2x and PBP1a encoded by mosaic genes is known to result in high cefotaxime resistance. In contrast, introduction of a mosaic pbp1a into the PBP2xT338G mutant did not lead to increased resistance. However, the mosaic PBP1a gene apparently complemented the PBP2xT338G defect, since Cia mutant derivatives grew normally. The data support the view that PBP2x and PBP1a interact with each other on some level and that alterations of both PBPs in resistant clinical isolates have evolved to ensure cooperation between both proteins

Persistence of the Chromosome End Regions at Low Copy Number in Mutant Strains of Streptomyces rimosus and Streptomyces lividans

Streptomycetes are important antibiotic producing bacteria that often exhibit genetic instability. One or both ends of the linear Streptomyces chromosome are lost spontaneously, resulting in viable mutant strains sometimes lacking hundreds of genes. We examined some strains of Streptomyces rimosus and Streptomyces lividans, which had been classified as »deletion mutants« and appeared to have lost chromosome end sequences. We discovered that the »deleted« sequences were still present in vegetative mycelium at a very low copy number so that they were normally not detected. The copy number in S. rimosus was estimated as 0.1–1.0 10–3/chromosome. Streptomyces spores contain the disappearing chromosome end sequences at a higher copy number than the vegetative mycelium, promoting their inheritance via spore preparations. This, in effect, represents a separation between germ line and deleted vegetative genomes, which has not been recognised before in Streptomyces, and has practical implications both for strain preservation and genetic studies

Streptococcus pneumoniae PBP2x mid-cell localization requires the C-terminal PASTA domains and is essential for cell shape maintenance

The transpeptidase activity of the essential penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae is believed to be important for murein biosynthesis required for cell division. To study the molecular mechanism driving localization of PBP2x in live cells, we constructed a set of N-terminal GFP-PBP2x fusions under the control of a zinc-inducible promoter. The ectopic fusion protein localized at mid-cell. Cells showed no growth defects even in the absence of the genomic pbp2x, demonstrating that GFP-PBP2x is functional. Depletion of GFP-PBP2x resulted in severe morphological alterations, confirming the essentiality of PBP2x and demonstrating that PBP2x is required for cell division and not for cell elongation. A genetically or antibiotic inactivated GFP-PBP2x still localized at septal sites. Remarkably, the same was true for a GFP-PBP2x derivative containing a deletion of the central transpeptidase domain, although only in the absence of the protease/chaperone HtrA. Thus localization is independent of the catalytic transpeptidase domain but requires the C-terminal PASTA domains, identifying HtrA as targeting GFP-PBP2x derivatives. Finally, PBP2x was positioned at the septum similar to PBP1a and the PASTA domain containing StkP protein, confirming that PBP2x is a key element of the divisome complex

Mechanism of β-Lactam Action in Streptococcus pneumoniae: the Piperacillin Paradox.

International audienceThe human pathogen Streptococcus pneumoniae has been fought for decades with β-lactam antibiotics. Resistance is now widespread, mediated by the expression of mosaic variants of the target enzymes, the penicillin-binding proteins or PBPs. Understanding the mode of action of β-lactams, not only in molecular details, but also in their physiological consequences, will be crucial to improve these drugs and counter resistance. In this work, we investigate the piperacillin paradox, by which this β-lactam selects primarily variants of PBP2b, whereas its most reactive target is PBP2x. These PBPs are both essential mono-functional transpeptidases involved in peptidoglycan assembly. PBP2x participates to septal synthesis, while PBP2b functions in peripheral elongation. The formation of "lemon"-shaped cells induced by piperacillin treatment is consistent with the inhibition of PBP2x. Following the examination of treated and untreated cells by electron microscopy, localization of the PBPs by epifluorescence microscopy, and determination of the inhibition time-course of the different PBPs, we propose a model of peptidoglycan assembly that can account for the piperacillin paradox