Structure of the essential peptidoglycan amidotransferase MurT/GatD complex from Streptococcus pneumoniae.

The universality of peptidoglycan in bacteria underlies the broad spectrum of many successful antibiotics. However, in our times of widespread resistance, the diversity of peptidoglycan modifications offers a variety of new antibacterials targets. In some Gram-positive species such as Streptococcus pneumoniae, Staphylococcus aureus, or Mycobacterium tuberculosis, the second residue of the peptidoglycan precursor, D-glutamate, is amidated into iso-D-glutamine by the essential amidotransferase MurT/GatD complex. Here, we present the structure of this complex at 3.0 Å resolution. MurT has central and C-terminal domains similar to Mur ligases with a cysteine-rich insertion, which probably binds zinc, contributing to the interface with GatD. The mechanism of amidation by MurT is likely similar to the condensation catalyzed by Mur ligases. GatD is a glutaminase providing ammonia that is likely channeled to the MurT active site through a cavity network. The structure and assay presented here constitute a knowledge base for future drug development studies.

CHiC, a new tandem affinity tag for the protein purification toolbox.

In the present work we have constructed a new tandem affinity purification tag and used it to purify two different polypeptides, PcsB and ECL1 from Streptococcus pneumoniae. PcsB probably functions as a peptidoglycan hydrolase and is believed to be involved in splitting of the septum during cell division. ECL1 is the extracellular domain of the membrane spanning protein FtsX. Experimental evidence indicates that the ECL1 domain controls the activity of PcsB through direct interaction (Sham et al., 2011). The affinity tag consists of an N-terminal 6xHis-tag, a choline binding domain followed by a proteolytic site specific for the TEV (tobacco etch virus) endopeptidase. Based on the choline-binding His-tag combination the new 16.5 kDa tag was named CHiC. CHiC-tagged PcsB and ECL1 were expressed in Escherichia coli and sequentially purified by employing diethylaminoethyl-cellulose affinity chromatography and Ni(2+) immobilized metal affinity chromatography. After TEV digestion, the CHiC-tag, TEV-protease and undigested fusion protein were easily separated from the target protein in a single purification step. By using this method, 4-7 mg of recombinant PcsB and ECL1 were obtained from one liter of cell culture with a purity estimated to be at least 95%. In addition, we found that the tag has the potential to function as a solubilisation partner as it markedly increased the solubility of PcsB. In sum, the CHiC-tag is a versatile tool that allows purification of milligram quantities of highly purified recombinant protein in only one or two steps.

Peptide-regulated gene depletion system developed for use in Streptococcus pneumoniae.

To facilitate the study of pneumococcal genes that are essential for viability or normal cell growth, we sought to develop a tightly regulated, titratable gene depletion system that interferes minimally with normal cellular functions. A possible candidate for such a system is the recently discovered signal transduction pathway regulating competence for natural transformation in Streptococcus thermophilus. This pathway, which is unrelated to the ComCDE pathway used for competence regulation in Streptococcus pneumoniae, has not been fully elucidated, but it is known to include a short unmodified signaling peptide, ComS*, an oligopeptide transport system, Ami, and a transcriptional activator, ComR. The transcriptional activator is thought to bind to an inverted repeat sequence termed the ECom box. We introduced the ComR protein and the ECom box into the genome of S. pneumoniae R6 and demonstrated that addition of synthetic ComS* peptide induced the transcription of a luciferase gene inserted downstream of the ECom box. To determine whether the ComRS system could be used for gene depletion studies, the licD1 gene was inserted behind the chromosomally located ECom box promoter by using the Janus cassette. Then, the native versions of licD1 and licD2 were deleted, and the resulting mutant was recovered in the presence of ComS*. Cultivation of the licD1 licD2 double mutant in the absence of ComS* gradually affected its ability to grow and propagate, demonstrating that the ComRS system functions as intended. In the present study, the ComRS system was developed for use in S. pneumoniae. In principle, however, it should work equally well in many other Gram-positive species.

New insights into the pneumococcal fratricide: relationship to clumping and identification of a novel immunity factor.

In 1971, Tomasz and Zanati discovered that competent pneumococci have a tendency to form aggregates when pelleted by centrifugation and resuspended in 0.01 N HCl by brief vortexing. Interestingly, no clumping was observed with parallel cultures of non-competent cells treated in the same way. We set out to elucidate the mechanism behind this striking phenomenon, and were able to show that it depends on extracellular DNA that is presumably released by so-called competence-induced cell lysis. Competence-induced cell lysis, which was first described a few years ago, seems to rely on the concerted action of several murein hydrolases. Our results confirmed and extended previous findings by showing that competence-induced aggregation is abolished in a lytA-lytC double mutant, and absolutely requires CbpD and its N-terminal CHAP amidase domain. Furthermore, we discovered a novel competence stimulating peptide (CSP)-induced immunity protein, encoded by the early competence gene comM (spr1762), which protects competent pneumococci against their own lysins. Together, the murein hydrolases and the immunity protein constitutes a CSP-controlled mechanism that allows competent pneumococci to commit fratricide by killing non-competent pneumococci sharing the same ecological niche. Through such predatory behaviour, pneumococci can get access to transforming DNA and nutrients, promote the release of virulence factors, and at the same time get rid of competitors.

Extracellular-peptide control of competence for genetic transformation in Streptococcus pneumoniae.

Bacteria, which often are subjected to fluctuations in nutrients, temperature, radiation, pH, etc., adapt to the physico-chemical environment they live in by making the appropriate changes in their gene expression patterns. During the last decades it has become increasingly clear that bacteria, in addition, have a "social life", and that changes in gene expression can also be elicited by the presence of other bacteria. Traditionally bacteria have been viewed as solitary organisms that in general do not interact with other bacteria in a coordinated manner. Recent advances in the field of bacterial cell-to-cell communication has proved this to be a misconception, and mounting evidence now show that bacterial group behaviour is ubiquitous in nature. Competence for natural genetic transformation in Streptococcus pneumoniae, which has been studied for more than seventy years, has become a paradigm for intercellular communication and cell density dependent regulation of gene expression in Gram-positive bacteria. There has been rapid progress recently in elucidating the molecular mechanisms behind regulation of natural competence in S. pneumoniae. In this review, we describe the current status of our knowledge of natural competence in this bacterium, with particular emphasis on the early phase of competence induction.