Identification of a CTX-M-255 ß-lactamase containing a G239S substitution selectively conferring resistance to penicillin/ß-lactamase inhibitor combinations.

OBJECTIVES: An Escherichia coli isolate, WGS1363, showed resistance to piperacillin/tazobactam but susceptibility to cephalosporins and contained a previously unrecognized ß-lactamase, CTX-M-255, as the only acquired ß-lactamase. CTX-M-255 was identical to CTX-M-27 except for a G239S substitution. Here, we characterize the hydrolytic spectrum of CTX-M-255 and a previously reported ß-lactamase, CTX-M-178, also containing a G239S substitution and compare it to their respective parental enzymes, CTX-M-27 and CTX-M-15. METHODS: All ß-lactamase genes were expressed in E. coli TOP10 and MICs to representative ß-lactam-antibiotics were determined. Furthermore, blaCTX-M-15,  blaCTX-M-27, blaCTX-M-178 and blaCTX-M-255 with C-terminal His-tag fusions were affinity purified for enzyme kinetic assays determining Michaelis-Menten kinetic parameters against representative ß-lactam-antibiotics and IC50s of clavulanate, sulbactam, tazobactam and avibactam. RESULTS: TOP10-transformants expressing blaCTX-M-178 and blaCTX-M-255 showed resistance to penicillin/ß-lactamase combinations and susceptibility to cephalothin and cefotaxime in contrast to transformants expressing blaCTX-M-15 and blaCTX-M-27. Determination of enzyme kinetic parameters showed that CTX-M-178 and CTX-M-255 both lacked hydrolytic activity against cephalosporins and showed impaired hydrolytic efficiency against penicillin antibiotics compared to their parental enzymes. Both enzymes appeared more active against piperacillin compared to benzylpenicillin and ampicillin. Compared to their parental enzymes, IC50s of ß-lactamase-inhibitors were increased more than 1000-fold for CTX-M-178 and CTX-M-255. CONCLUSIONS: CTX-M-178 and CTX-M-255, both containing a G239S substitution, conferred resistance to piperacillin/tazobactam and may be characterized as inhibitor-resistant CTX-M ß-lactamases. Inhibitor resistance was accompanied by loss of activity against cephalosporins and monobactams. These findings add to the necessary knowledge base for predicting antibiotic susceptibility from genotypic data.

GGDEF domain as spatial on-switch for a phosphodiesterase by interaction with landmark protein HubP.

In bacteria, the monopolar localization of enzymes and protein complexes can result in a bimodal distribution of enzyme activity between the dividing cells and heterogeneity of cellular behaviors. In Shewanella putrefaciens, the multidomain hybrid diguanylate cyclase/phosphodiesterase PdeB, which degrades the secondary messenger c-di-GMP, is located at the flagellated cell pole. Here, we show that direct interaction between the inactive diguanylate cyclase (GGDEF) domain of PdeB and the FimV domain of the polar landmark protein HubP is crucial for full function of PdeB as a phosphodiesterase. Thus, the GGDEF domain serves as a spatially controlled on-switch that effectively restricts PdeBs activity to the flagellated cell pole. PdeB regulates abundance and activity of at least two crucial surface-interaction factors, the BpfA surface-adhesion protein and the MSHA type IV pilus. The heterogeneity in c-di-GMP concentrations, generated by differences in abundance and timing of polar appearance of PdeB, orchestrates the population behavior with respect to cell-surface interaction and environmental spreading.

Corrigendum: The Stand-Alone PilZ-Domain Protein MotL Specifically Regulates the Activity of the Secondary Lateral Flagellar System in Shewanella putrefaciens.

[This corrects the article DOI: 10.3389/fmicb.2021.668892.].

The Stand-Alone PilZ-Domain Protein MotL Specifically Regulates the Activity of the Secondary Lateral Flagellar System in Shewanella putrefaciens.

A number of bacterial species control the function of the flagellar motor in response to the levels of the secondary messenger c-di-GMP, which is often mediated by c-di-GMP-binding proteins that act as molecular brakes or clutches to slow the motor rotation. The gammaproteobacterium Shewanella putrefaciens possesses two distinct flagellar systems, the primary single polar flagellum and a secondary system with one to five lateral flagellar filaments. Here, we identified a protein, MotL, which specifically regulates the activity of the lateral, but not the polar, flagellar motors in response to the c-di-GMP levels. MotL only consists of a single PilZ domain binding c-di-GMP, which is crucial for its function. Deletion and overproduction analyses revealed that MotL slows down the lateral flagella at elevated levels of c-di-GMP, and may speed up the lateral flagellar-mediated movement at low c-di-GMP concentrations. In vitro interaction studies hint at an interaction of MotL with the C-ring of the lateral flagellar motors. This study shows a differential c-di-GMP-dependent regulation of the two flagellar systems in a single species, and implicates that PilZ domain-only proteins can also act as molecular regulators to control the flagella-mediated motility in bacteria.

The small DUF1127 protein CcaF1 from Rhodobacter sphaeroides is an RNA-binding protein involved in sRNA maturation and RNA turnover.

Many different protein domains are conserved among numerous species, but their function remains obscure. Proteins with DUF1127 domains number >17 000 in current databases, but a biological function has not yet been assigned to any of them. They are mostly found in alpha- and gammaproteobacteria, some of them plant and animal pathogens, symbionts or species used in industrial applications. Bioinformatic analyses revealed similarity of the DUF1127 domain of bacterial proteins to the RNA binding domain of eukaryotic Smaug proteins that are involved in RNA turnover and have a role in development from Drosophila to mammals. This study demonstrates that the 71 amino acid DUF1127 protein CcaF1 from the alphaproteobacterium Rhodobacter sphaeroides participates in maturation of the CcsR sRNAs that are processed from the 3' UTR of the ccaF mRNA and have a role in the oxidative stress defense. CcaF1 binds to many cellular RNAs of different type, several mRNAs with a function in cysteine / methionine / sulfur metabolism. It affects the stability of the CcsR RNAs and other non-coding RNAs and mRNAs. Thus, the widely distributed DUF1127 domain can mediate RNA-binding, affect stability of its binding partners and consequently modulate the bacterial transcriptome, thereby influencing different physiological processes.

The GGDEF Domain of the Phosphodiesterase PdeB in Shewanella putrefaciens Mediates Recruitment by the Polar Landmark Protein HubP.

Bacteria commonly exhibit a high degree of cellular organization and polarity which affect many vital processes such as replication, cell division, and motility. In Shewanella and other bacteria, HubP is a polar marker protein which is involved in proper chromosome segregation, placement of the chemotaxis system, and various aspects of pilus- and flagellum-mediated motility. Here, we show that HubP also recruits a transmembrane multidomain protein, PdeB, to the flagellated cell pole. PdeB is an active phosphodiesterase and degrades the second messenger c-di-GMP. In Shewanella putrefaciens, PdeB affects both the polar and the lateral flagellar systems at the level of function and/or transcription in response to environmental medium conditions. Mutant analysis on fluorescently labeled PdeB indicated that a diguanylate cyclase (GGDEF) domain in PdeB is strictly required for HubP-dependent localization. Bacterial two-hybrid and in vitro interaction studies on purified proteins strongly indicate that this GGDEF domain of PdeB directly interacts with the C-terminal FimV domain of HubP. Polar localization of PdeB occurs late during the cell cycle after cell division and separation and is not dependent on medium conditions. In vitro activity measurements did not reveal a difference in PdeB phosphodiesterase activities in the presence or absence of the HubP FimV domain. We hypothesize that recruitment of PdeB to the flagellated pole by HubP may create an asymmetry of c-di-GMP levels between mother and daughter cells and may assist in organization of c-di-GMP-dependent regulation within the cell.IMPORTANCE c-di-GMP-dependent signaling affects a range of processes in many bacterial species. Most bacteria harbor a plethora of proteins with domains which are potentially involved in synthesis and breakdown of c-di-GMP. A potential mechanism to elicit an appropriate c-di-GMP-dependent response is to organize the corresponding proteins in a spatiotemporal fashion. Here, we show that a major contributor to c-di-GMP levels and flagellum-mediated swimming in Shewanella, PdeB, is recruited to the flagellated cell pole by the polar marker protein HubP. Polar recruitment involves a direct interaction between HubP and a GGDEF domain in PdeB, demonstrating a novel mechanism of polar targeting by the widely conserved HubP/FimV polar marker.