Gram-negative bacteria rely on an asymmetric outer membrane for barrier integrity, with phospholipids confined to the inner leaflet and lipopolysaccharide or lipooligosaccharide (LOS) forming the outer leaflet. Although these glycolipids were long considered essential, recent findings challenge this view. Here, using Acinetobacter baumannii, we identify lipid asymmetry as a structural checkpoint controlling access to LOS-independent survival. Disruption of phospholipid transport and degradation destabilizes membrane balance, creating a permissive state that enables the emergence of LOS-deficient, colistin-resistant variants. Lipidomic and transcriptomic analyses reveal coordinated envelope remodeling, including altered peptidoglycan synthesis and enhanced envelope trafficking. Loss of LOS coincides with repression of PBP1A, and maintaining its activity blocks adaptation. We propose a three-state model—basal, permissive, and adapted—that explains how envelope architecture governs antibiotic resistance and membrane remodeling.

The multilayered cell envelope of Acinetobacter baumannii is an essential structure that maintains cellular integrity and protects the bacterial cell against external stresses and antibiotics. It consists of an inner membrane, a thin peptidoglycan (PG) layer and an asymmetric outer membrane (OM) enriched in lipooligosaccharide (LOS), whose lipid A moiety is the target of colistin, a last-resource antibiotic. Although lipid A is essential in most Gram-negatives, A. baumannii can survive without LOS through envelope remodeling, particularly in strains producing low levels of the bifunctional penicillin-binding protein PBP1A (encoded by mrcA ) or in D mrcA mutants. Here, we identify a functional interplay between the LD-transpeptidase LdtJ, which generates 3-3 cross-links, and PBP1A, which catalyzes 4-3 transpeptidation during PG synthesis. We show that simultaneous inactivation of both enzymes severely affected growth, viability, morphology, and OM homeostasis. PG analyses revealed that the Δ ldtJ Δ mrcA mutants displays reduced overall cross-linkage and shorter glycan chains, producing a weakened sacculus. Co-immunoprecipitation demonstrated that PBP1A associates with LdtJ, supporting their coordinated activity at sites of PG synthesis. Notably, Δ ldtJ Δ mrcA mutants exhibited the highest recovery frequency of colistin-resistant, LOS-deficient variants compared with wild type or single mutants. Together, our findings demonstrate that coupling between 4-3 and 3-3 transpeptidation is critical for envelope stability in A. baumannii and highlight how disrupting this coordination favors the emergence of colistin resistance. This work identifies a conserved PG remodeling vulnerability that directly links PG integrity to the evolution of antibiotic resistance, offering a new conceptual framework for destabilizing the A. baumannii envelope.

The gram-negative cell envelope is a critical interface between the bacterium and its environment, serving as a selective barrier for nutrient uptake and defense against harmful agents. It also facilitates environmental sensing and adaptive responses. Structurally, it comprises the outer membrane, inner membrane, and periplasmic space, which contains the peptidoglycan layer—a conserved polymer that maintains cell shape and withstands internal turgor pressure. Peptidoglycan consists of glycan strands connected by short peptides, forming a mesh-like structure. In gram-negative bacteria, most peptidoglycan subunits contain tetrapeptides, generated by DD-carboxypeptidases (DD-CPases) that cleave the terminal D-alanine from pentapeptides. Although gram-negative bacteria encode multiple DD-CPases, their precise roles in maintaining cell shape and structural integrity remain poorly understood. The nosocomial pathogen Acinetobacter baumannii encodes three putative DD-CPases. To investigate their functions, we generated single and double mutants in dacC, dacD, and pbpG, which encode homologs of Escherichia coli DD-CPases PBP5 and PBP6a, PBP6b, and the endopeptidase PBP7, respectively. We assessed the mutants for changes in cell morphology, growth dynamics, and pH-dependent stress tolerance. Additionally, we analyzed their peptidoglycan composition to determine the biochemical consequences of enzyme inactivation. Each mutant showed distinct alterations in coccobacillary morphology and growth. Peptidoglycan analysis showed DD-CPase activity, with PBP6b also exhibiting endopeptidase activity. Together, our results demonstrate that each peptidoglycan-modifying enzyme contributes uniquely to cell growth, morphology, and pH tolerance, underscoring their non-redundant functions.IMPORTANCEDD-peptidases, including carboxypeptidases and endopeptidases, are crucial for maintaining cell envelope homeostasis, with distinct roles for each enzyme in cell wall biogenesis and structural integrity. The enzymatic characterization presented in this study not only advances our understanding of fundamental Acinetobacter baumannii biology but also highlights these enzymatic activities as targets for the development of innovative therapeutic strategies to combat infections caused by this multidrug-resistant microbe.

Regulation of class A penicillin-binding proteins (aPBPs) in peptidoglycan biosynthesis is incompletely understood in Gram-positive bacteria. One example is activation of aPBP2a by GpsB and phosphorylated MacP in the ovoid-shaped pathogen, Streptococcus pneumoniae. We set out to examine whether phosphorylation of Thr residues other than Thr32 contributed to MacP activation of aPBP2a. We also wanted to determine whether GpsB and MacP activation of aPBP2a were related. Here we report that MacP was phosphorylated about equally at Thr32 and Thr56 in physiological and biochemical assays. However, based on transformation and growth assays, phosphorylation of MacP was not required for aPBP2a activation. A structure-function analysis confirmed that most of the MacP cytoplasmic domain, which was predicted by AlphaFold3 to be disordered, was not required for aPBP2a activation. These analyses further identified amino acids in the MacP transmembrane domain and the aPBP2a juxtamembrane region, as well as a variant of the GpsB-binding motif in the membrane-proximal cytoplasmic region of MacP, required for aPBP2a activation. Together, these results support a tripartite model in which GpsB acts as an adapter for activation of aPBP2a by MacP. Finally, additional interaction, Tn-seq, and growth assays suggested other modes of direct or indirect regulation of aPBP2a activity.

Background/Objectives: The management of urinary tract infections (UTIs) has become an increasingly challenging medical intervention. This study explores whether adoption of a precision medicine model could improve the management of acute uncomplicated pyelonephritis (uAPN) or complicated UTIs (cUTIs) compared with the standard of care approach, in hospitalized patients. Methods: From January 2022 to March 2024, all patients affected by uAPN or cUTIs and attending our urological institution were randomized to receive the following: antibiotic treatment according to guidelines and recommendations (standard of care group) or antibiotic treatment according to the precision medical model (intervention group). The main outcome measures were the rates of clinical success and the length of hospitalization. The time until switching to oral treatment was regarded as a secondary outcome measure. Results: Eighty-three patients were enrolled in the standard of care group, while seventy-nine patients were enrolled in the intervention group. While the overall clinical success rate was similar in the two groups (75 vs. 72; p = 0.97), a statistically significant difference was observed between the two groups in terms of length of hospitalization (8 days vs. 5 days; p = 0.03) and time to switch to oral treatment (96 h vs. 72 h; p = 0.04). A statistically significant difference was found between the two groups regarding the need to change antimicrobial therapy during hospitalization [12 out of 80 vs. 6 out of 77; p = 0.04]. Conclusions: Adoption of the precision medicine model appears as a valuable means to improve the management of patients with uAPN and cUTIs. By reducing the period of hospitalization and the time to switch to oral treatment, the precision medicine model also improves antimicrobial stewardship in the management of UTIs.

OBJECTIVES:To evaluate the feasibility of a novel point-of-care test (POCT) management strategy including phase contrast microscopy for bacteriuria and urinary dipsticks for erythrocytes to guide antibiotic prescribing in women with suspected uncomplicated urinary tract infection (uUTI) in general practice. DESIGN:Pilot cluster randomised controlled trial in 20 general practices in Germany. Practices were assigned 1:1 to POCT-guided management or usual care. All urine samples were sent for urine culture. Follow-up over 28 days involved symptom diaries, telephone interviews, and medical record review. OUTCOMES:Primary outcomes were recruitment and retention rates. Secondary outcomes included total and inappropriate antibiotic use, symptom duration and burden, recurrent and upper UTIs, re-consultations, and diagnostic accuracy of microscopy versus urine culture. Mixed-effects models accounted for clustering. RESULTS:Over 8 months, 157 women were recruited (90 intervention, 67 control), median of 7.5 patients per practice (range 1-15). Participant retention at day 28 was 75%. Baseline characteristics were well balanced. Antibiotic use was similar in both groups: 77% (intervention) vs. 79% (control) at initial consultation. The mean number of antibiotic courses over 28 days was 0.96 (intervention) vs. 1.00 (control), with no indication of reduced prescribing. Phase-contrast microscopy showed limited diagnostic accuracy, especially for ruling out infection (negative predictive value 46%). Exploratory analyses suggested that if GPs had access to urine culture results at the point of care, antibiotic prescribing in the intervention group could have been higher than in routine care. CONCLUSION:The POCT-guided management approach for suspected uUTIs is feasible but presents implementation and methodological challenges. Recruitment varied across sites and was lower in the control group practices, highlighting the risk of differential recruitment. Retention was below the expected 80%, indicating the need for efficient follow-up strategies in future trials. Explorative analyses suggest that simply adding diagnostic information may not support antibiotic stewardship. Novel POCTs should be carefully assessed for their influence on prescribing before routine use. Trial registration: ClinicalTrials.gov NCT05667207.

INTRODUCTION:Uncomplicated urinary tract infections (uUTIs) in women are common infections encountered in primary care. Evidence suggests that rapid point-of-care tests (POCTs) to detect bacteria and erythrocytes in urine at presentation may help primary care clinicians to identify women with uUTIs in whom antibiotics can be withheld without influencing clinical outcomes. This pilot study aims to provide preliminary evidence on whether a POCT informed management of uUTI in women can safely reduce antibiotic use.METHODS AND ANALYSIS:This is an open-label two-arm parallel cluster-randomised controlled pilot trial. 20 general practices affiliated with the Bavarian Practice-Based Research Network (BayFoNet) in Germany were randomly assigned to deliver patient management based on POCTs or to provide usual care. POCTs consist of phase-contrast microscopy to detect bacteria and urinary dipsticks to detect erythrocytes in urine samples. In both arms, urine samples will be obtained at presentation for POCTs (intervention arm only) and microbiological analysis. Women will be followed-up for 28 days from enrolment using self-reported symptom diaries, telephone follow-up and a review of the electronic medical record. Primary outcomes are feasibility of patient enrolment and retention rates per site, which will be summarised by means and SDs, with corresponding confidence and prediction intervals. Secondary outcomes include antibiotic use for UTI at day 28, time to symptom resolution, symptom burden, number of recurrent and upper UTIs and re-consultations and diagnostic accuracy of POCTs versus urine culture as the reference standard. These outcomes will be explored at cluster-levels and individual-levels using descriptive statistics, two-sample hypothesis tests and mixed effects models or generalised estimation equations.ETHICS AND DISSEMINATION:The University of Würzburg institutional review board approved MicUTI on 16 December 2022 (protocol n. 109/22-sc). Study findings will be disseminated through peer-reviewed publications, conferences, reports addressed to clinicians and the local citizen's forums.TRIAL REGISTRATION NUMBER:ClinicalTrials.gov NCT05667207.

Urinary tract infections (UTIs) are highly prevalent and frequently treated with antibiotics, making antimicrobial stewardship (AMS) crucial in this field to benefit both individual patients and society. This narrative review examines recent advancements in managing urinary tract infections, focusing on AMS and antimicrobial resistance (AMR). AMS is essential in urology, where antibiotics are frequently prescribed for UTI. AMS promotes optimal antibiotic use to improve patient outcomes, control AMR, and safeguard public health. Key AMS practices in urology include selecting antibiotics at the appropriate dose and duration, especially for uncomplicated UTIs, asymptomatic bacteriuria, and catheter-associated infections. Following evidence-based guidelines, such as those from EAU and the IDSA, helps prioritize narrow-spectrum antibiotics and discourage empirical broad-spectrum use. Regular audits and feedback on antibiotic use help align practices with AMS goals, reducing inappropriate prescriptions. Multidisciplinary AMS teams, including urologists, microbiologists, and pharmacists, enhance treatment precision and antibiotic optimization in complex cases. Reassessing antibiotic therapy based on culture results after 48-72 h enables clinicians to refine treatment, minimizing unnecessary broad-spectrum use and strengthening AMS efforts in urology. In conclusion, addressing AMR in urology requires a careful approach that combines evidence-based antibiotic use, attention to local resistance patterns, and patient-specific factors. Non-antibiotic strategies for recurrent UTI prevention, judicious catheter management, and tailored treatment for complex infections are key AMS components. Emerging technologies in diagnostics and precision medicine offer tools for targeted, personalized therapy, enhancing AMS efforts, and helping to reduce reliance on broad-spectrum antibiotics.

BACKGROUND:Even if Meares-Stamey 4-glass (M&S) test is regarded a decisive tool for diagnosing prostatitis its use is only rarely performed in everyday clinical practice. Here, we analyze if the diagnostic yield of the M&S test could be improved by a pre-test categorization of patients due to undergo a M&S test. METHODS:All clinical and microbiological data of patients who underwent M&S test in two urological centers from January 2004 to December 2021 were analyzed in this retrospective cohort study. One center has a dedicated staff member for the study of prostatitis (Cohort I), while the other center is a general urological unit (Cohort II). All patients were divided into 3 groups on the basis of the assembled data: patients with symptoms related to prostatitis only (Group I), patients with symptoms related to both prostatitis and BPH (Group II), patients with symptoms related to BPH only (Group III). The rates of positive microbiological results in each group were compared. RESULTS:In the whole period, 9347 patients were analyzed and categorized as follows: Group I, 1884; Group II, 5151; Group III, 2312. Three-thousand and eight-hundred twenty-three patients showed positive culture results (40.9%). The most common isolated species was Escherichia coli (49.7%), followed by Enteroccus spp. (31.8%). The rates of positive M&S tests in the different symptom groups were: Group I, 1532 (81.4%); Group II, 1494 (29.0%); Group III, 797 (34.4%). The overall rate of positive M&S tests in each urology center showed that the center with a staff member who is dedicated to prostatitis studies (Cohort I) had a significantly higher rate of positive M&S tests than the general urological department (Cohort II) (64.3% vs 31.4%; p < 0.001). CONCLUSIONS:Symptom-based patient selection and dedicated staff members will increase the diagnostic yield of the M&S test and reduce the number of unnecessary tests.

StkP, the Ser/Thr protein kinase of the major human pathogen Streptococcus pneumoniae, monitors cell wall signals and regulates growth and division in response. In vivo, StkP interacts with GpsB, a cell division protein required for septal ring formation and closure, that affects StkP-dependent phosphorylation. Here, we report that although StkP has basal intrinsic kinase activity, GpsB promotes efficient autophosphorylation of StkP and phosphorylation of StkP substrates. Phosphoproteomic analyzes showed that GpsB is phosphorylated at several Ser and Thr residues. We confirmed that StkP directly phosphorylates GpsB in vitro and in vivo, with T79 and T83 being the major phosphorylation sites. In vitro, phosphoablative GpsB substitutions had a lower potential to stimulate StkP activity, whereas phosphomimetic substitutions were functional in terms of StkP activation. In vivo, substitutions of GpsB phosphoacceptor residues, either phosphoablative or mimetic, had a negative effect on GpsB function, resulting in reduced StkP-dependent phosphorylation and impaired cell division. The bacterial two-hybrid assay and co-immunoprecipitation of GpsB from cells with differentially active StkP indicated that increased phosphorylation of GpsB resulted in a more efficient interaction of GpsB with StkP. Our data suggest that GpsB acts as an adaptor that directly promotes StkP activity by mediating interactions within the StkP signaling hub, ensuring StkP recruitment into the complex and substrate specificity. We present a model that interaction of StkP with GpsB and its phosphorylation and dephosphorylation dynamically modulate kinase activity during exponential growth and under cell wall stress of S. pneumoniae, ensuring the proper functioning of the StkP signaling pathway.

Frequently touched surfaces (FTS) that are contaminated with pathogens are one of the main sources of nosocomial infections, which commonly include hospital-acquired and healthcare-associated infections (HAIs). HAIs are considered the most common adverse event that has a significant burden on the public’s health worldwide currently. The persistence of pathogens on contaminated surfaces and the transmission of multi-drug resistant (MDR) pathogens by way of healthcare surfaces, which are frequently touched by healthcare workers, visitors, and patients increase the risk of acquiring infectious agents in hospital environments. Moreover, not only in hospitals but also in high-traffic public places, FTS play a major role in the spreading of pathogens. Consequently, attention has been devoted to developing novel and alternative methods to tackle this problem. This study planned to produce and characterize innovative functionalized enameled coated surfaces supplemented with 1% AgNO3 and 2% AgNO3. Thus, the antimicrobial properties of the enamels against relevant nosocomial pathogens including the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli and the yeast Candida albicans were assessed using the ISO:22196:2011 norm.

GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen Streptococcus pneumoniae (pneumococcus; Spn) and other low‐GC Gram‐positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP(Spn) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications (≈21–176 genes) containing murZ (MurZ‐family homolog of MurA) or murA that suppress ΔgpsB or ΔstkP. These duplications arose from three different repeated sequences and demonstrate the facility of pneumococcus to modulate gene dosage of numerous genes. Overproduction of MurZ or MurA alone or overproduction of MurZ caused by ΔkhpAB mutations suppressed ΔgpsB or ΔstkP phenotypes to varying extents. ΔgpsB and ΔstkP were also suppressed by MurZ amino‐acid changes distant from the active site, including one in commonly studied laboratory strains, and by truncation or deletion of the homolog of IreB(ReoM). Unlike in other Gram‐positive bacteria, MurZ is predominant to MurA in pneumococcal cells. However, ΔgpsB and ΔstkP were not suppressed by ΔclpCP, which did not alter MurZ or MurA amounts. These results support a model in which regulation of MurZ and MurA activity, likely by IreB(Spn), is the only essential requirement for StkP‐mediated protein phosphorylation in exponentially growing D39 pneumococcal cells.

GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen Streptococcus pneumoniae (pneumococcus; Spn ) and other low-GC Gram-positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP( Spn ) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications (≈21-176 genes) containing murZ (MurZ-family homolog of MurA) or murA that suppress Δ gpsB or Δ stkP . These duplications arose from three different repeated sequences and demonstrate the facility of pneumococcus to modulate gene dosage of numerous genes. Overproduction of MurZ or MurA alone or overexpression of MurZ caused by Δ khpAB mutations suppressed Δ gpsB or Δ stkP phenotypes to varying extents. Δ gpsB and Δ stkP were also suppressed by MurZ amino-acid changes distant from the active site, including one in commonly studied laboratory strains, and by truncation or deletion of the homolog of IreB(ReoM). Unlike in other Gram-positive bacteria, MurZ is predominant to MurA in pneumococcal cells. However, Δ gpsB and Δ stkP were not suppressed by Δ clpCP , which did not alter MurZ or MurA amounts. These results support a model in which regulation of MurZ and MurA activity, likely by IreB( Spn ), is the only essential requirement for protein phosphorylation in exponentially growing D39 pneumococcal cells.

The reversibility of bacterial resistance to antibiotics is poorly understood. Therefore, the aim of this study was to determine, over a period of five years, the effect of fluoroquinolone (FQ) use in primary care on the development and gradual decay of Escherichia coli resistance to FQ. In this matched case–control study, we linked three sources of secondary data of the Health Service of the Autonomous Province of Bolzano, Italy. Cases were all those with an FQ-resistant E. coli (QREC)-positive culture from any site during a 2016 hospital stay. Data were analyzed using conditional logistic regression. A total of 409 cases were matched to 993 controls (FQ-sensitive E. coli) by the date of the first isolate. Patients taking one or more courses of FQ were at higher risk of QREC colonization/infection. The risk was highest during the first year after FQ was taken (OR 2.67, 95%CI 1.92–3.70, p < 0.0001), decreased during the second year (OR 1.54, 95%CI 1.09–2.17, p = 0.015) and became undetectable afterwards (OR 1.09, 95%CI 0.80–1.48, p = 0.997). In the first year, the risk of resistance was highest after greater cumulative exposure to FQs. Moreover, older age, male sex, longer hospital stays, chronic obstructive pulmonary disease (COPD) and diabetes mellitus were independent risk factors for QREC colonization/infection. A single FQ course significantly increases the risk of QREC colonization/infection for no less than two years. This risk is higher in cases of multiple courses, longer hospital stays, COPD and diabetes; in males; and in older patients. These findings may inform public campaigns and courses directed to prescribers to promote rational antibiotic use.

•Secondary infections and progression of COVID-19;.•Clinical laboratory data are crucial to monitoring COVID-19 progression;.•Microbiological data are fundamental for the early identification of secondary infections and best antibiotic treatment.

Microbial secondary infections can contribute to an increase in the risk of mortality in COVID-19 patients, particularly in case of severe diseases. In this study, we collected and evaluated the clinical, laboratory and microbiological data of COVID-19 critical ill patients requiring intensive care (ICU) to evaluate the significance and the prognostic value of these parameters. One hundred seventy-eight ICU patients with severe COVID-19, hospitalized at the S. Francesco Hospital of Nuoro (Italy) in the period from March 2020 to May 2021, were enrolled in this study. Clinical data and microbiological results were collected. Blood chemistry parameters, relative to three different time points, were analyzed through multivariate and univariate statistical approaches. Seventy-four percent of the ICU COVID-19 patients had a negative outcome, while 26% had a favorable prognosis. A correlation between the laboratory parameters and days of hospitalization of the patients was observed with significant differences between the two groups. Moreover, Staphylococcus aureus, Enterococcus faecalis, Candida spp, Pseudomonas aeruginosa and Klebsiella pneumoniae were the most frequently isolated microorganisms from all clinical specimens. Secondary infections play an important role in the clinical outcome. The analysis of the blood chemistry tests was found useful in monitoring the progression of COVID-19.

The emergence and spreading of the SARS-CoV-2 pandemic has forced the focus of attention on a significant issue: the realization of antimicrobial surfaces for public spaces, which do not require extensive use of disinfectants. Silver represents one of the most used elements in this context, thanks to its excellent biocidal performance. This work describes a simple method for the realization of anodized aluminum layers, whose antimicrobial features are ensured by the co-deposition with silver nitrate. The durability and the chemical resistance of the samples were evaluated by means of several accelerated degradation tests, such as the exposure in a salt spray chamber, the contact with synthetic sweat and the scrub test, highlighting the residual influence of silver in altering the protective behavior of the alumina layers. Furthermore, the ISO 22196:2011 standard was used as the reference protocol to set up an assay to measure the effective antibacterial activity of the alumina-Ag layers against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, even at low concentrations of silver. Finally, the Ag-containing aluminum oxide layers exhibited excellent antimicrobial performances also following the chemical–physical degradation processes, ensuring good durability over time of the antimicrobial surfaces. Overall, this work introduces a simple route for the realization of anodized aluminum surfaces with excellent antibacterial properties.

Porcelain enamel coatings are silica-based inorganic coatings, which are successfully applied to different kind of metallic substrates in applications where high chemical resistance and corrosion protection are needed. In addition, these coatings meet very modern criteria of durability and environmental sustainability. For this reason, they could be a valid solution for application in healthcare settings and public spaces, where cleaning and disinfection are frequently required. The possibility to produce durable antibacterial enamel coatings would broaden their application areas and represent a possible solution to address the spread of pathogens in many public spaces. The main of this work is to develop innovative antibacterial enamel coatings with the addition of silver-based additives (AgNO3) and test their antibacterial efficacy in operando conditions (i.e., after chemical and mechanical degradation) by exploiting antibacterial assays according to the ISO 22196:2011 standard. Three different sample types will be taken under consideration: the reference sample, and the sample with the addition of 1 wt% AgNO3, and the sample with the addition of 2 wt% AgNO3. The surface properties and the micro-structure of the samples will be deeply investigated by surface sensitive techniques, optical microscopy, and electron microscopy observations. The antimicrobial efficacy of these samples will be tested against Escherichia coli and Staphylococcus aureus both before and after chemical/physiochemical degradation.

Acinetobacter baumannii is a Gram-negative pathogen, known to acquire resistance to antibiotics used in the clinic. The RNA-binding proteome of this bacterium is poorly characterized, in particular for what concerns the proteins containing RNA Recognition Motif (RRM). Here, we browsed the A. baumannii proteome for homologous proteins to the human HuR(ELAVL1), an RNA binding protein containing three RRMs. We identified a unique locus that we called AB-Elavl, coding for a protein with a single RRM with an average of 34% identity to the first HuR RRM. We also widen the research to the genomes of all the bacteria, finding 227 entries in 12 bacterial phyla. Notably we observed a partial evolutionary divergence between the RNP1 and RNP2 conserved regions present in the prokaryotes in comparison to the metazoan consensus sequence. We checked the expression at the transcript and protein level, cloned the gene and expressed the recombinant protein. The X-ray and NMR structural characterization of the recombinant AB-Elavl revealed that the protein maintained the typical β1α1β2β3α2β4 and three-dimensional organization of eukaryotic RRMs. The biochemical analyses showed that, although the RNP1 and RNP2 show differences, it can bind to AU-rich regions like the human HuR, but with less specificity and lower affinity. Therefore, we identified an RRM-containing RNA-binding protein actually expressed in A. baumannii.

RodZ of rod-shaped bacteria functions to link MreB filaments to the Rod peptidoglycan (PG) synthase complex that moves circumferentially perpendicular to the long cell axis, creating hoop-like sidewall PG. Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus; Spn) that lack MreB, use a different modality for peripheral PG elongation that emanates from the midcell of dividing cells. Yet, S. pneumoniae encodes a RodZ homolog similar to RodZ in rod-shaped bacteria. We show here that the helix-turn-helix and transmembrane domains of RodZ(Spn) are essential for growth at 37°C. ΔrodZ mutations are suppressed by Δpbp1a, mpgA(Y488D), and ΔkhpA mutations that suppress ΔmreC, but not ΔcozE. Consistent with a role in PG elongation, RodZ(Spn) co-localizes with MreC and aPBP1a throughout the cell cycle and forms complexes and interacts with PG elongasome proteins and regulators. Depletion of RodZ(Spn) results in aberrantly shaped, non-growing cells and mislocalization of elongasome proteins MreC, PBP2b, and RodA. Moreover, Tn-seq reveals that RodZ(Spn), but not MreCD(Spn), displays a specific synthetic-viable genetic relationship with aPBP1b, whose function is unknown. We conclude that RodZ(Spn) acts as a scaffolding protein required for elongasome assembly and function and that aPBP1b, like aPBP1a, plays a role in elongasome regulation and possibly peripheral PG synthesis.