No abstract

Summaryβ β β β -Lactamases, which evolved from bacterial penicillin-binding proteins (PBPs) involved in peptidoglycan (PG) synthesis, confer resistance to β β β β -lactam antibiotics. While investigating the genetic basis of biofilm development by Pseudomonas aeruginosa , we noted that plasmid vectors encoding the common β β β β -lactamase marker TEM-1 caused defects in twitching motility (mediated by type IV pili), adherence and biofilm formation without affecting growth rates. Similarly, strains of Escherichia coli carrying TEM-1-encoding vectors grew normally but showed reduced adherence and biofilm formation, showing this effect was not species-specific. Introduction of otherwise identical plasmid vectors carrying tetracycline or gentamicin resistance markers had no effect on biofilm formation or twitching motility. The effect is restricted to class A and D enzymes, because expression of the class D Oxa-3 β β β β -lactamase, but not class B or C β β β β -lactamases, impaired biofilm formation by E. coli and P. aeruginosa . Site-directed mutagenesis of the catalytic Ser of TEM-1, but not Oxa-3, abolished the biofilm defect, while disruption of either TEM-1 or Oxa-3 expression restored wild-type levels of biofilm formation. We hypothesized that the A and D classes of β β β β -lactamases, which are related to low molecular weight (LMW) PBPs, may sequester or alter the PG substrates of such enzymes and interfere with normal cell wall turnover. In support of this hypothesis, deletion of the E. coli LMW PBPs 4, 5 and 7 or combinations thereof, resulted in cumulative defects in biofilm formation, similar to those seen in β β β β -lactamase-expressing transformants. Our results imply that horizontal acquisition of β β β β -lactamase resistance enzymes can have a phenotypic cost to bacteria by reducing their ability to form biofilms. β β β β -Lactamases likely affect PG remodelling, manifesting as perturbation of structures involved in bacterial adhesion that are required to initiate biofilm formation.

In bacteria, several physiological processes once thought to be the products of uniformly dispersed reactions are now known to be highly asymmetric, with some exhibiting interesting geometric localizations. In particular, the cell envelope of Escherichia coli displays a form of subcellular differentiation in which peptidoglycan and outer membrane proteins at the cell poles remain stable for generations while material in the lateral walls is diluted by growth and turnover. To determine if material in the side walls was organized in any way, we labeled outer membrane proteins with succinimidyl ester-linked fluorescent dyes and then grew the stained cells in the absence of dye. Labeled proteins were not evenly dispersed in the envelope but instead appeared as helical ribbons that wrapped around the outside of the cell. By staining the O8 surface antigen of E. coli 2443 with a fluorescent derivative of concanavalin A, we observed a similar helical organization for the lipopolysaccharide (LPS) component of the outer membrane. Fluorescence recovery after photobleaching indicated that some of the outer membrane proteins remained freely diffusible in the side walls and could also diffuse into polar domains. On the other hand, the LPS O antigen was virtually immobile. Thus, the outer membrane of E. coli has a defined in vivo organization in which a subfraction of proteins and LPS are embedded in stable domains at the poles and along one or more helical ribbons that span the length of this gram-negative rod.

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxyterminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal ␤-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core.Escherichia coli expresses four low-molecular-weight (LMW) DD-carboxypeptidase penicillin binding proteins (PBPs) that share considerable nucleic acid sequence identity (PBPs 4, 5, and 6 and DacD), suggesting that they diverged from a common primordial enzyme (16). The classic explanation for this apparent redundancy is that the DD-carboxypeptidases can modify peptidoglycan in similar ways so that they serve as auxiliaries of one another (3). However, arguing against this idea is the observation that PBP 5 plays a predominate role among the LMW PBPs in maintaining the normal morphology of E. coli, because the loss of this protein severely alters the diameter, contour, and topology of mutants lacking multiple PBPs (5,12,18,19). Thus, among the LMW PBPs, PBP 5 must have unique properties that allow the protein to modify bacterial shape.There are at least two structural differences among the DDcarboxypeptidases that might explain how PBP 5 contributes to uniform cell shape and why the homologous enzymes are not equivalent substitutes. First, differences may exist in the amphipathic carboxy terminus that is proposed to anchor each enzyme to the outer face of the cytoplasmic membrane (6,13,14,21). Variations among the DD-carboxypeptidase anchors might affect protein localization, enzymatic activity, or interactions with ot...