Biology subjects

Havarstein, L. S.

Publications and source records attributed to Havarstein, L. S..

7 recordsLinked to original sources

bioRxiv2025

LytF contributes to pilus extrusion during natural competence in Streptococcus sanguinis SK36.

Streptococci may enter a physiological state called competence, during which they express a specific set of genes required for exogenous DNA uptake and its subsequent integration into the genome through homologous recombination. This process, termed natural transformation, facilitates the horizontal acquisition of genetic material, potentially conferring adaptive advantages that enhance bacterial survival under selective pressures. To make homologous DNA available in the surrounding environment, Streptococcus pneumoniae expresses a cell wall hydrolase (CbpD) that lyses and kills closely related species. This process has been coined fratricide, and the acting hydrolase a fratricin. A significant fraction of streptococcal species does not encode a CbpD-like protein, but another competence-induced peptidoglycan hydrolase LytF. It has been speculated that LytF serves the same purpose as CbpD, however, our investigations into the role of LytF in Streptococcus sanguinis revealed no evidence supporting LytF as a fratricin. Instead, we show that LytF is involved in natural transformation by promoting DNA uptake. An essential part of DNA uptake is the competence-induced type IV pilus, which facilitates DNA uptake by pulling nearby DNA toward the cell. By immunoblotting and microscopy imaging, we found that LytF increases the extracellular levels of the major pilus component ComGC, suggesting that LytF may modify peptidoglycan to promote pilus extrusion across the cell wall, thereby enhancing the efficiency of DNA uptake. ImportanceStreptococci are a significant cause of severe infections in both humans and animals. They are particularly adept at acquiring new genes through horizontal gene transfer as they can become competent for natural transformation. This allows them to quickly adapt to selective pressure and spread genes involved in virulence and antibiotic resistance. In Streptococcus sanguinis, the competence-induced peptidoglycan hydrolase LytF has been reported to stimulate natural transformation. Our study adds to the understanding of this process by demonstrating that LytF promotes extrusion of the transformation pilus required for DNA uptake.

microbiology↗

bioRxiv2022

Competence remodels the pneumococcal cell wall providing resistance to fratricide and surface exposing key virulence factors

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation and virulence. Competent bacteria produce so called fratricins such as CbpD, that kill non-competent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall- and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

microbiology↗

bioRxiv2020

EloR interacts with the lytic transglycosylase MltG at midcell in Streptococcus pneumoniae R6.

The ellipsoid shape of Streptococcus pneumoniae is determined by the synchronized actions of the elongasome and the divisome, which have the task of creating a protective layer of peptidoglycan (PG) enveloping the cell membrane. The elongasome is necessary for expanding PG in the longitudinal direction whereas the divisome synthesizes the PG that divides one cell into two. Although there is still little knowledge about how these two modes of PG synthesis are coordinated, it was recently discovered that two RNA-binding proteins called EloR and KhpA are part of a novel regulatory pathway controlling elongation in S. pneumoniae. EloR and KhpA form a complex that work closely with the Ser/Thr kinase StkP to regulate cell elongation. Here, we have further explored how this regulation occur. EloR/KhpA is found at midcell, a localization fully dependent on EloR. Using a bacterial two-hybrid assay we probed EloR against several elongasome proteins and found an interaction with the lytic transglycosylase homolog MltG. By using EloR as bait in immunoprecipitation assays, MltG was pulled down confirming that they are part of the same protein complex. Fluorescent microscopy demonstrated that the Jag domain of EloR is essential for EloRs midcell localization and its interaction with MltG. Since MltG is found at midcell independent of EloR, our results suggest that MltG is responsible for recruitment of the EloR/KhpA complex to the division zone to regulate cell elongation. ImportanceBacterial cell division has been a successful target for antimicrobial agents for decades. How different pathogens regulate cell division is, however, poorly understood. To fully exploit the potential for future antibiotics targeting cell division, we need to understand the details of how the bacteria regulate and construct cell wall during this process. Here we have revealed that the newly identified EloR/KhpA complex, regulating cell elongation in S. pneumoniae, forms a complex with the essential peptidoglycan transglycosylase MltG at midcell. EloR, KhpA and MltG are conserved among many bacterial species and the EloR/KhpA/MltG regulatory pathway is most likely a common mechanism employed by many Gram-positive bacteria to coordinate cell elongation and septation.

microbiology↗

bioRxiv2020

Structural characterization of the essential cell division protein FtsE and its interaction with FtsX in Streptococcus pneumoniae

FtsEX is a membrane complex widely conserved across diverse bacterial genera and involved in critical processes such as recruitment of division proteins and in spatial and temporal regulation of muralytic activity during cell division or sporulation. FtsEX is a member of the ABC transporter superfamily, where FtsX is an integral membrane protein and FtsE is an ATPase, required for mechanotransmission of the signal from the cytosol through the membrane, to regulate the activity of cell-wall hydrolases in the periplasm. Both proteins are essential in the major human respiratory pathogenic bacterium, Streptococcus pneumoniae and interact with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report the high-resolution structures of pneumococcal FtsE in complex with different nucleotides. Structural analysis reveals that FtsE contains all the conserved structural motifs associated with ATPase activity, and allowed interpretation of the in vivo dimeric arrangement in both ADP and ATP states. Interestingly, three specific FtsE regions were identified with high structural plasticity that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for FtsE contact, were identified and validated by in vivo mutagenesis studies showing that this interaction is essential for cell growth and proper morphology. IMPORTANCEBacterial cell division is a central process that requires exquisite orchestration of both the cell wall biosynthetic and lytic machineries. The essential membrane complex FtsEX, widely conserved across bacteria, play a central role by recruiting proteins to the divisome apparatus and by regulating periplasmic muralytic activity from the cytosol. FtsEX is a member of the Type VII family of the ABC-superfamily but instead transporter, couple ATP hydrolysis by FtsE to mechanically transduce a conformational signal to activate PG hydrolases. So far, no structural information is available for FtsE. Here we provide the structural characterization of FtsE confirming its ATPase nature and revealing regions with high structural plasticity key for FtsX binding. The complementary region in FtsX has been also identified and validated in vivo. Our results provide evidences on how difference between ATP and ADP states in FtsE would dramatically alter FtsEX interaction with PG hydrolase PcsB in pneumococcal division.

biochemistry↗

bioRxiv2019

Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall.

In oval shaped Streptococcus pneumoniae, septal and longitudinal peptidoglycan synthesis is performed by independent functional complexes; the divisome and the elongasome. Penicillin binding proteins (PBPs) were long considered as the key peptidoglycan synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with non-PBP glycosyltransferases (FtsW and RodA) to make up the core peptidoglycan synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD to show that class A PBPs have an autonomous role during cell wall synthesis in S. pneumoniae. Purified CbpD was shown to target the septum of S. pneumoniae cells. Using assays to specifically inhibit PBP2x, we demonstrate that CbpD specifically target nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes or repairs nascent peptidoglycan synthesized by FtsW/PBP2x. Our results support a model in which pneumococcal peptidoglycan is made by three functional entities, the divisome, the elongasome and a peptidoglycan-repairing or -remodelling complex consisting of bifunctional PBPs. To our knowledge this is the first time a specific function has been identified for class A PBPs in bacterial cell wall synthesis.

microbiology↗

bioRxiv2018

Structure of the large extracellular loop of FtsX and its interaction with the essential peptidoglycan hydrolase PcsB in Streptococcus pneumoniae

Streptococcus pneumoniae is a leading killer of infants and immunocompromised adults and has become increasingly resistant to major antibiotics. Therefore, the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically targeting essential proteins for the synthesis and breakdown of peptidoglycan. One complex important to this process is FtsEX. FtsEX comprises an integral membrane protein (FtsX) and cytoplasmic ATPase (FtsE) that resembles an ATP-binding cassette (ABC) transporter. Here, we present NMR solution structural and crystallographic models of the large extracellular domain of FtsX, denoted ECL1. The structure of ECL1 reveals an upper extended {beta}-hairpin and a lower -helical lobe, each extending from a mixed -{beta} core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB, via the coiled-coil domain of PcsB (PcsB-CC). Characterization of S. pneumoniae D39 derived strains harboring mutations in the -helical lobe shows that this subdomain is essential for cell viability and required for proper cell division of S. pneumoniae.\n\nIMPORTANCEFtsX is a ubiquitous bacterial integral membrane protein involved in cell division that regulates the activity of peptidoglycan (PG) hydrolases. FtsX is representative of a large group of ABC3 superfamily proteins that function as \"mechanotransmitters,\" proteins that relay signals from inside to the outside of the cell. Here we present a structural characterization of the large extracellular loop (ECL1) of FtsX from the human opportunistic pathogen Streptococcus pneumoniae. We show a direct interaction between the peptidoglycan hydrolase PcsB and FtsX, and demonstrate that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics.

microbiology↗

bioRxiv2018

CozEa and CozEb play overlapping and essential roles in controlling cell division in Staphylococcus aureus

Staphylococcus aureus needs to control the position and timing of cell division and cell wall synthesis to maintain its spherical shape. We identified two membrane proteins, named CozEa and CozEb, which together are important for proper cell division in S. aureus. CozEa and CozEb are homologs of the cell elongation regulator CozESpn of Streptococcus pneumoniae. While cozEa and cozEb were not essential individually, the {Delta}cozEa{Delta}cozEb double mutant was lethal. To study the functions of cozEa and cozEb, we constructed a CRISPR interference (CRISPRi) system for S. aureus, allowing transcriptional knockdown of essential genes. CRISPRi knockdown of cozEa in the {Delta}cozEb strain (and vice versa) causes cell morphological defects and aberrant nucleoid staining, showing that cozEa and cozEb have overlapping functions and are important for normal cell division. We found that CozEa and CozEb interact with the cell division protein EzrA, and that EzrA-GFP mislocalizes in the absence of CozEa and CozEb. Furthermore, the CozE-EzrA interaction is conserved in S. pneumoniae, and cell division is mislocalized in cozESpn-depleted S. pneumoniae cells. Together, our results show that CozE proteins mediate control of cell division in S. aureus and S. pneumoniae, likely via interactions with key cell division proteins such as EzrA.

microbiology↗