Mechanisms of Methicillin Resistance in Staphylococcus aureus.

Staphylococcus aureus is a major human and veterinary pathogen worldwide. Methicillin-resistant S. aureus (MRSA) poses a significant and enduring problem to the treatment of infection by such strains. Resistance is usually conferred by the acquisition of a nonnative gene encoding a penicillin-binding protein (PBP2a), with significantly lower affinity for ß-lactams. This resistance allows cell-wall biosynthesis, the target of ß-lactams, to continue even in the presence of typically inhibitory concentrations of antibiotic. PBP2a is encoded by the mecA gene, which is carried on a distinct mobile genetic element (SCCmec), the expression of which is controlled through a proteolytic signal transduction pathway comprising a sensor protein (MecR1) and a repressor (MecI). Many of the molecular and biochemical mechanisms underlying methicillin resistance in S. aureus have been elucidated, including regulatory events and the structure of key proteins. Here we review recent advances in this area.

Growth and Division of the Peptidoglycan Matrix.

Most bacteria are surrounded by a peptidoglycan cell wall that defines their shape and protects them from osmotic lysis. The expansion and division of this structure therefore plays an integral role in bacterial growth and division. Additionally, the biogenesis of the peptidoglycan layer is the target of many of our most effective antibiotics. Thus, a better understanding of how the cell wall is built will enable the development of new therapies to combat the rise of drug-resistant bacterial infections. This review covers recent advances in defining the mechanisms involved in assembling the peptidoglycan layer with an emphasis on discoveries related to the function and regulation of the cell elongation and division machineries in the model organisms Escherichia coli and Bacillus subtilis.

A conserved zinc-binding site in Acinetobacter baumannii PBP2 required for elongasome-directed bacterial cell shape.

Acinetobacter baumannii is a gram-negative bacterial pathogen that causes challenging nosocomial infections. ß-lactam targeting of penicillin-binding protein (PBP)-mediated cell wall peptidoglycan (PG) formation is a well-established antimicrobial strategy. Exposure to carbapenems or zinc (Zn)-deprived growth conditions leads to a rod-to-sphere morphological transition in A. baumannii, an effect resembling that caused by deficiency in the RodA-PBP2 PG synthesis complex required for cell wall elongation. While it is recognized that carbapenems preferentially acylate PBP2 in A. baumannii and therefore block the transpeptidase function of the RodA-PBP2 system, the molecular details underpinning cell wall elongation inhibition upon Zn starvation remain undefined. Here, we report the X-ray crystal structure of A. baumannii PBP2, revealing an unexpected Zn coordination site in the transpeptidase domain required for protein stability. Mutations in the Zn-binding site of PBP2 cause a loss of bacterial rod shape and increase susceptibility to ß-lactams, therefore providing a direct rationale for cell wall shape maintenance and Zn homeostasis in A. baumannii. Furthermore, the Zn-coordinating residues are conserved in various ß- and γ-proteobacterial PBP2 orthologs, consistent with a widespread Zn-binding requirement for function that has been previously unknown. Due to the emergence of resistance to virtually all marketed antibiotic classes, alternative or complementary antimicrobial strategies need to be explored. These findings offer a perspective for dual inhibition of Zn-dependent PG synthases and metallo-ß-lactamases by metal chelating agents, considered the most sought-after adjuvants to restore ß-lactam potency against gram-negative bacteria.

Genomic diversity and antimicrobial susceptibility of invasive Neisseria meningitidis in South Africa, 2016-2021.

BACKGROUND: Invasive meningococcal isolates in South Africa have in previous years (<2008) been characterized by serogroup B, C, W and Y lineages over time, with penicillin intermediate resistance (peni) at 6%. We describe the population structure and genomic markers of peni among invasive meningococcal isolates in South Africa, 2016-2021. METHODS: Meningococcal isolates were collected through national, laboratory-based invasive meningococcal disease (IMD) surveillance. Phenotypic antimicrobial susceptibility testing and whole-genome sequencing were performed, and the mechanism of reduced penicillin susceptibility was assessed in silico. RESULTS: Of 585 IMD cases reported during the study period, culture and PCR-based capsular group was determined for 477/585 (82%); and 241/477 (51%) were sequenced. Predominant serogroups included NmB (210/477; 44%), NmW (116/477; 24%), NmY (96/477; 20%) and NmC (48/477; 10%). Predominant clonal complexes (CC) were CC41/44 in NmB (27/113; 24%), CC11 in NmW (46/56; 82%), CC167 in NmY (23/44; 53%), and CC865 in NmC (9/24; 38%). Peni was detected in 16% (42/262) of isolates, and was due to the presence of a penA mosaic, with the majority harboring penA7, penA9 or penA14. CONCLUSION: IMD lineages circulating in South Africa were consistent with those circulating prior to 2008, however peni was higher than previously reported, and occurred in a variety of lineages.

Structural and kinetic analysis of the monofunctional Staphylococcus aureus PBP1.

Staphylococcus aureus, an ESKAPE pathogen, is a major clinical concern due to its pathogenicity and manifold antimicrobial resistance mechanisms. The commonly used ß-lactam antibiotics target bacterial penicillin-binding proteins (PBPs) and inhibit crosslinking of peptidoglycan strands that comprise the bacterial cell wall mesh, initiating a cascade of effects leading to bacterial cell death. S. aureus PBP1 is involved in synthesis of the bacterial cell wall during division and its presence is essential for survival of both antibiotic susceptible and resistant S. aureus strains. Here, we present X-ray crystallographic data for S. aureus PBP1 in its apo form as well as acyl-enzyme structures with distinct classes of ß-lactam antibiotics representing the penicillins, carbapenems, and cephalosporins, respectively: oxacillin, ertapenem and cephalexin. Our structural data suggest that the PBP1 active site is readily accessible for substrate, with little conformational change in key structural elements required for its covalent acylation of ß-lactam inhibitors. Stopped-flow kinetic analysis and gel-based competition assays support the structural observations, with even the weakest performing ß-lactams still having comparatively high acylation rates and affinities for PBP1. Our structural and kinetic analysis sheds insight into the ligand-PBP interactions that drive antibiotic efficacy against these historically useful antimicrobial targets and expands on current knowledge for future drug design and treatment of S. aureus infections.

Clinical isolates of Streptococcus mitis/oralis-related species with reduced carbapenem susceptibility, harboring amino acid substitutions in penicillin-binding proteins in Japan.

Streptococcus mitis/oralis group isolates with reduced carbapenem susceptibility have been reported, but its isolation rate in Japan is unknown. We collected 356 clinical α-hemolytic streptococcal isolates and identified 142 of them as S. mitis/oralis using partial sodA sequencing. The rate of meropenem non-susceptibility was 17.6% (25/142). All 25 carbapenem-non-susceptible isolates harbored amino acid substitutions in/near the conserved motifs in PBP1A, PBP2B, and PBP2X. Carbapenem non-susceptibility is common among S. mitis/oralis group isolates in Japan.

Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi-enzyme complexes in Escherichia coli.

The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh-like sacculus requires the combined activity of peptidoglycan synthases and hydrolases. In Escherichia coli, the activity of two PG synthases is driven by lipoproteins anchored in the outer membrane (OM). However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the OM lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and division biosynthetic complexes based on its localization and genetic interactions. Consistent with such a role, we reconstitute PG multi-enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that peptidoglycan regulators and adaptors are part of PG biosynthetic multi-enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases.

Structural and biochemical analysis of penicillin-binding protein 2 from Campylobacter jejuni.

Penicillin-binding protein 2 (PBP2) plays a key role in the formation of peptidoglycans in bacterial cell walls by crosslinking glycan chains through transpeptidase activity. PBP2 is also found in Campylobacter jejuni, a pathogenic bacterium that causes food-borne enteritis in humans. To elucidate the essential structural features of C. jejuni PBP2 (cjPBP2) that mediate its biological function, we determined the crystal structure of cjPBP2 and assessed its protein stability under various conditions. cjPBP2 adopts an elongated two-domain structure, consisting of a transpeptidase domain and a pedestal domain, and contains typical active site residues necessary for transpeptidase activity, as observed in other PBP2 proteins. Moreover, cjPBP2 responds to ß-lactam antibiotics, including ampicillin, cefaclor, and cefmetazole, suggesting that ß-lactam antibiotics inactivate cjPBP2. In contrast to typical PBP2 proteins, cjPBP2 is a rare example of a Zn2+-binding PBP2 protein, as the terminal structure of its transpeptidase domain accommodates a Zn2+ ion via three cysteine residues and one histidine residue. Zn2+ binding helps improve the protein stability of cjPBP2, providing opportunities to develop new C. jejuni-specific antibacterial drugs that counteract the Zn2+-binding ability of cjPBP2.

The peptidoglycan synthase PBP interacts with PLASTID DIVISION2 to promote chloroplast division in Physcomitrium patens.

The peptidoglycan (PG) layer, a core component of the bacterial cell wall, has been retained in the Physcomitrium patens chloroplasts. The PG layer entirely encompasses the P. patens chloroplast, including the division site, but how PG biosynthesis cooperates with the constriction of two envelope membranes at the chloroplast division site remains elusive. Here, focusing on the PG synthase penicillin-binding protein (PBP), we performed cytological and molecular analyses to dissect the mechanism of chloroplast division in P. patens. We showed that PBP, acting in the final step of PG biosynthesis, is likely a chloroplast inner envelope protein that can aggregate at mid-chloroplasts during chloroplast division. Physcomitrium patens had five orthologs of PLASTID DIVISION2 (PDV2), an outer envelope component of the chloroplast division complex. Our data indicated that PpPDV2 proteins interact with PpPBP and are responsible for recruiting PpPBP to the chloroplast division site, in addition to PpDRP5B. Furthermore, we found that PBP deletion and carbenicillin application restrain constriction of the chloroplast division complex, rather than its assembly. This work provides direct molecular evidence for a link between chloroplast division of P. patens and PG biosynthesis and indicates that PG biosynthesis is required for the constriction of the chloroplast division apparatus in P. patens.

Characterization of Ceftriaxone-Resistant Haemophilus influenzae Among Korean Children.

BACKGROUND: Haemophilus influenzae is a frequently encountered pathogen responsible for respiratory tract infections in children. Following the detection of ceftriaxone-resistant H. influenzae at our institution, we aimed to investigate the resistance mechanisms of ceftriaxone in H. influenzae, with a particular focus on alterations in penicillin-binding protein 3 (PBP3) and ß-lactamase production. METHODS: Among H. influenzae isolates collected at Asan Medical Center Children's Hospital from March 2014 to April 2019, ceftriaxone-resistant strains by the disk-diffusion test were included. Ceftriaxone minimum inhibitory concentrations (MICs) were determined using the E-test according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The presence of ß-lactamase was assessed through cefinase test and TEM-1/ROB-1 polymerase chain reaction (PCR). PBP3 alterations were explored via ftsI gene sequencing. RESULTS: Out of the 68 collected strains, 21 exhibited resistance to ceftriaxone in disk diffusion tests. Two strains were excluded due to failed subculture. Among 19 ceftriaxone-resistant H. influenzae isolates, eighteen were non-typeable H. influenzae, and twelve were positive for TEM-1 PCR. Isolates were classified into groups II (harboring only N526K, n = 3), III (N526K+S385T, n = 2), III+ (S385T+L389F+N526K, n = 11), and III-like+ (S385T+L389F+R517H, n = 3) according to the PBP3 alteration pattern. With a median ceftriaxone MIC of 0.190 mg/L (range, 0.008-0.750), the median ceftriaxone MIC was the highest in group III-like+ (0.250 mg/L), followed by groups III+ (0.190 mg/L), III (0.158 mg/L), and II (0.012 mg/L). All three strains belonging to group II, which did not harbor the S385T substitution, had ceftriaxone MICs of ≤ 0.125 mg/L. CONCLUSION: The emergence of ceftriaxone-resistant H. influenzae with ceftriaxone MIC values of up to 0.75 mg/L was observed even in children in South Korea, with most associated with S385T and L389F substitutions. The N526K mutation alone does not significantly impact ceftriaxone resistance. Further large-scale studies are essential to investigate changes in antibiotic resistance patterns and factors influencing antibiotic resistance in H. influenzae isolated from pediatric patients in Korea.

Structural Insights into the Penicillin-Binding Protein 4 (DacB) from Mycobacterium tuberculosis.

Mycobacterium tuberculosis, a major cause of mortality from a single infectious agent, possesses a remarkable mycobacterial cell envelope. Penicillin-Binding Proteins (PBPs) are a family of bacterial enzymes involved in the biosynthesis of peptidoglycan. PBP4 (DacB) from M. tuberculosis (MtbPBP4) has been known to function as a carboxypeptidase, and the role and significance of carboxypeptidases as targets for anti-tuberculosis drugs or antibiotics have been extensively investigated over the past decade. However, their precise involvement remains incompletely understood. In this study, we employed predictive modeling and analyzed the three-dimensional structure of MtbPBP4. Interestingly, MtbPBP4 displayed a distinct domain structure compared to its homologs. Docking studies with meropenem verified the presence of active site residues conserved in PBPs. These findings establish a structural foundation for comprehending the molecular function of MtbPBP4 and offer a platform for the exploration of novel antibiotics.

Molecular Basis of Influence of A501X Mutations in Penicillin-Binding Protein 2 of Neisseria gonorrhoeae Strain 35/02 on Ceftriaxone Resistance.

The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k2/KS, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k2, and the weakening of the binding affinity, i.e., an increase in the substrate constant, KS. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2A501V variant exhibits a higher k2/KS value, whereas for PBP2A501R and PBP2A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k2/KS changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the ß-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k2/KS value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the ß3-ß4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k2/KS value. Thus, the experimentally observed decrease in the k2/KS in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k2, and the increase in KS.

Penicillin-Binding Proteins and Alternative Dual-Beta-Lactam Combinations for Serious Enterococcus faecalis Infections with Elevated Penicillin MICs.

Ampicillin-ceftriaxone has become a first-line therapy for Enterococcus faecalis endocarditis. We characterized the penicillin-binding protein (PBP) profiles of various E. faecalis strains and tested for synergy to better inform beta-lactam options for the treatment of E. faecalis infections. We assessed the affinity of PBP2B from elevated-MIC strain E. faecalis LS4828 compared to type strain JH2-2 using the fluorescent beta-lactam Bocillin FL. We also characterized pbp4 and pbpA structures and PBP4 and PBP2B expression and used deletion and complementation studies to assess the impact of PBP2B on the levels of resistance. We tested penicillin-susceptible and -resistant E. faecalis isolates against ceftriaxone or ceftaroline combinations with other beta-lactams in 24-h time-kill studies. Two penicillin-susceptible strains (JH2-2 and L2052) had identical pbp sequences and similar PBP expression levels. One reduced-penicillin-susceptibility strain (L2068) had pbp sequences identical to those of the susceptible strains but expressed more PBP4. The second decreased-penicillin-susceptibility strain (LS4828) had amino acid substitutions in both PBP4 and PBP2B and expressed increased quantities of both proteins. PBP2B did not appear to contribute significantly to the elevated beta-lactam MICs. No synergy was demonstrable against the strains with both mutated PBPs and increased expression (L2068 and LS4828). Meropenem plus ceftriaxone or ertapenem plus ceftriaxone demonstrated the most consistent synergistic activity. PBP2B of strain LS4828 does not contribute significantly to reduced penicillin susceptibility. Neither the MIC nor the level of PBP expression correlated directly with the identified synergistic combinations when tested at static subinhibitory concentrations.

Unraveling the mechanism of ceftaroline-induced allosteric regulation in penicillin-binding protein 2a: insights for novel antibiotic development against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) acquires high-level resistance against ß-lactam antibiotics by expressing penicillin-binding protein 2a (PBP2a). PBP2a is a cell wall-synthesizing protein whose closed active site exhibits a reduced binding affinity toward ß-lactam antibiotics. Ceftaroline (CFT), a fifth-generation cephalosporin, can effectively inhibit the PBP2a activity by binding to an allosteric site to trigger the active site opening, allowing a second CFT to access the active site. However, the essential mechanism behind the allosteric behavior of PBP2a remains unclear. Herein, computational simulations are employed to elucidate how CFT allosterically regulates the conformation and dynamics of the active site of PBP2a. While CFT stabilizes the allosteric domain surrounding it, it simultaneously enhances the dynamics of the catalytic domain. Specifically, the study successfully captured the opening process of the active pocket in the allosteric CFT-bound systems and discovered that CFT alters the potential signal-propagating pathways from the allosteric site to the active site. These findings reveal the implied mechanism of the CFT-mediated allostery in PBP2a and provide new insights into dual-site drug design or combination therapy against MRSA targeting PBP2a.

Activity of ETX0462 toward Some Burkholderia spp.

Burkholderia cepacia complex (Bcc) and Burkholderia gladioli are opportunistic human pathogens that are inherently multidrug resistant, limiting treatment options for infections. Here, a novel diazabicyclooctane, ETX0462, was evaluated for activity against Bcc and B. gladioli. Ninety-eight percent of the isolates examined in this study were susceptible. ETX0462 was found to demonstrate in vitro activity superior to that of currently available treatment options (e.g., trimethoprim-sulfamethoxazole and ceftazidime).

ß-Lactams from the Ocean.

The title of this essay is as much a question as it is a statement. The discovery of the ß-lactam antibiotics-including penicillins, cephalosporins, and carbapenems-as largely (if not exclusively) secondary metabolites of terrestrial fungi and bacteria, transformed modern medicine. The antibiotic ß-lactams inactivate essential enzymes of bacterial cell-wall biosynthesis. Moreover, the ability of the ß-lactams to function as enzyme inhibitors is of such great medical value, that inhibitors of the enzymes which degrade hydrolytically the ß-lactams, the ß-lactamases, have equal value. Given this privileged status for the ß-lactam ring, it is therefore a disappointment that the exemplification of this ring in marine secondary metabolites is sparse. It may be that biologically active marine ß-lactams are there, and simply have yet to be encountered. In this report, we posit a second explanation: that the value of the ß-lactam to secure an ecological advantage in the marine environment might be compromised by its close structural similarity to the ß-lactones of quorum sensing. The steric and reactivity similarities between the ß-lactams and the ß-lactones represent an outside-of-the-box opportunity for correlating new structures and new enzyme targets for the discovery of compelling biological activities.

A Review on Five and Six-Membered Heterocyclic Compounds Targeting the Penicillin-Binding Protein 2 (PBP2A) of Methicillin-Resistant Staphylococcus aureus (MRSA).

Staphylococcus aureus is a common human pathogen. Methicillin-resistant Staphylococcus aureus (MRSA) infections pose significant and challenging therapeutic difficulties. MRSA often acquires the non-native gene PBP2a, which results in reduced susceptibility to ß-lactam antibiotics, thus conferring resistance. PBP2a has a lower affinity for methicillin, allowing bacteria to maintain peptidoglycan biosynthesis, a core component of the bacterial cell wall. Consequently, even in the presence of methicillin or other antibiotics, bacteria can develop resistance. Due to genes responsible for resistance, S. aureus becomes MRSA. The fundamental premise of this resistance mechanism is well-understood. Given the therapeutic concerns posed by resistant microorganisms, there is a legitimate demand for novel antibiotics. This review primarily focuses on PBP2a scaffolds and the various screening approaches used to identify PBP2a inhibitors. The following classes of compounds and their biological activities are discussed: Penicillin, Cephalosporins, Pyrazole-Benzimidazole-based derivatives, Oxadiazole-containing derivatives, non-ß-lactam allosteric inhibitors, 4-(3H)-Quinazolinones, Pyrrolylated chalcone, Bis-2-Oxoazetidinyl macrocycles (ß-lactam antibiotics with 1,3-Bridges), Macrocycle-embedded ß-lactams as novel inhibitors, Pyridine-Coupled Pyrimidinones, novel Naphthalimide corbelled aminothiazoximes, non-covalent inhibitors, Investigational-ß-lactam antibiotics, Carbapenem, novel Benzoxazole derivatives, Pyrazolylpyridine analogues, and other miscellaneous classes of scaffolds for PBP2a. Additionally, we discuss the penicillin-binding protein, a crucial target in the MRSA cell wall. Various aspects of PBP2a, bacterial cell walls, peptidoglycans, different crystal structures of PBP2a, synthetic routes for PBP2a inhibitors, and future perspectives on MRSA inhibitors are also explored.

PBP1A Directly Interacts with the Divisome Complex to Promote Septal Peptidoglycan Synthesis in Acinetobacter baumannii.

The class A penicillin-binding proteins (aPBPs), PBP1A and PBP1B, are major peptidoglycan synthases that synthesize more than half of the peptidoglycan per generation in Escherichia coli. Whereas aPBPs have distinct roles in peptidoglycan biosynthesis during growth (i.e., elongation and division), they are semiredundant; disruption of either is rescued by the other to maintain envelope homeostasis and promote proper growth. Acinetobacter baumannii is a nosocomial pathogen that has a high propensity to overcome antimicrobial treatment. A. baumannii contains both PBP1A and PBP1B (encoded by mrcA and mrcB, respectively), but only mrcA deletion decreased fitness and contributed to colistin resistance through inactivation of lipooligosaccharide biosynthesis, indicating that PBP1B was not functionally redundant with the PBP1A activity. While previous studies suggested a distinct role for PBP1A in division, it was unknown whether its role in septal peptidoglycan biosynthesis was direct. Here, we show that A. baumannii PBP1A has a direct role in division through interactions with divisome components. PBP1A localizes to septal sites during growth, where it interacts with the transpeptidase PBP3, an essential division component that regulates daughter cell formation. PBP3 overexpression was sufficient to rescue the division defect in ΔmrcA A. baumannii; however, PBP1A overexpression was not sufficient to rescue the septal defect when PBP3 was inhibited, suggesting that their activity is not redundant. Overexpression of a major dd-carboxypeptidase, PBP5, also restored the canonical A. baumannii coccobacilli morphology in ΔmrcA cells. Together, these data support a direct role for PBP1A in A. baumannii division and highlights its role as a septal peptidoglycan synthase. IMPORTANCE Peptidoglycan biosynthesis is a validated target of ß-lactam antibiotics, and it is critical that we understand essential processes in multidrug-resistant pathogens such as Acinetobacter baumannii. While model systems such as Escherichia coli have shown that PBP1A is associated with side wall peptidoglycan synthesis, we show herein that A. baumannii PBP1A directly interacts with the divisome component PBP3 to promote division, suggesting a unique role for the enzyme in this highly drug-resistant nosocomial pathogen. A. baumannii demonstrated unanticipated resistance and tolerance to envelope-targeting antibiotics, which may be driven by rewired peptidoglycan machinery and may underlie therapeutic failure during antibiotic treatment.

Characterization of TseB: A new actor in cell wall elongation in Bacillus subtilis.

Penicillin-binding proteins (PBPs) are crucial enzymes of peptidoglycan assembly and targets of ß-lactam antibiotics. However, little is known about their regulation. Recently, membrane proteins were shown to regulate the bifunctional transpeptidases/glycosyltransferases aPBPs in some bacteria. However, up to now, regulators of monofunctional transpeptidases bPBPs have yet to be revealed. Here, we propose that TseB could be such a PBP regulator. This membrane protein was previously found to suppress tetracycline sensitivity of a Bacillus subtilis strain deleted for ezrA, a gene encoding a regulator of septation ring formation. In this study, we show that TseB is required for B. subtilis normal cell shape, tseB mutant cells being shorter and wider than wild-type cells. We observed that TseB interacts with PBP2A, a monofunctional transpeptidase. While TseB is not required for PBP2A activity, stability, and localization, we show that the overproduction of PBP2A is deleterious in the absence of TseB. In addition, we showed that TseB is necessary not only for efficient cell wall elongation during exponential phase but also during spore outgrowth, as it was also observed for PBP2A. Altogether, our results suggest that TseB is a new member of the elongasome that regulates PBP2A function during cell elongation and spore germination.

The NaHCO3-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus (MRSA) Is Influenced by mecA Genotype.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a leading cause of many invasive clinical syndromes, and pose treatment difficulties due to their in vitro resistance to most ß-lactams on standard laboratory testing. A novel phenotype frequently identified in MRSA strains, termed 'NaHCO3-responsiveness', is a property whereby strains are susceptible in vitro to many ß-lactams in the presence of NaHCO3. Specific mecA genotypes, repression of mecA/PBP2a expression and perturbed maturation of PBP2a by NaHCO3 have all been associated with this phenotype. The aim of this study was to define the relationship between specific mecA genotypes and PBP2a substitutions, on the one hand, with NaHCO3-responsiveness in vitro. Mutations were made in the mecA ribosomal binding site (RBS -7) and at amino acid position 246 of its coding region in parental strains MW2 (NaHCO3-responsive) and C36 (NaHCO3- nonresponsive) to generate 'swap' variants, each harboring the other's mecA-RBS/coding region genotypes. Successful swaps were confirmed by both sequencing, as well as predicted swap of in vitro penicillin-clavulanate susceptibility phenotypes. MW2 swap variants harboring the nonresponsive mecA genotypes became NaHCO3-nonresponsive (resistant to the ß-lactam, oxacillin [OXA]), in the presence of NaHCO3. Moreover, these swap variants had lost NaHCO3-mediated repression of mecA/PBP2a expression. In contrast, C36 swap variants harboring the NaHCO3-responsive mecA genotypes remained NaHCO3-nonresponsive phenotypically, and still exhibited nonrepressible mecA/PBP2a expression. These data demonstrate that in addition to the mecA genotype, NaHCO3-responsiveness may also depend on strain-specific genetic backgrounds.