- Volume 161, Issue 4, 2015

Penicillin-binding protein 5 (PBP5), a dd-carboxypeptidase, maintains cell shape and intrinsic beta-lactam resistance in E. coli. A strain lacking PBP5 loses intrinsic beta-lactam resistance and simultaneous lack of two other PBPs results in aberrantly shaped cells. PBP5 expression in trans complements the shape and restores the lost beta-lactam resistance. PBP5 has an ‘Ω-loop’-like region similar to that in class A beta-lactamases. It was previously predicted that Leu182 present in the ‘Ω-like’ loop of PBP5 corresponds to Glu166 in PER-1 beta-lactamase. Here, we studied the physiological and biochemical effects of the Leu182Glu mutation in PBP5. Upon overexpression in septuple PBP mutants, ~75 % of cells were abnormally shaped and intrinsic beta-lactam resistance maintenance was partially lost. Biochemically, the purified soluble mutated PBP5 (smPBP5) retained low acylation ability for penicillin. The turnover number of smPBP5 for artificial and peptidoglycan mimetic substrates was ~10-fold less than that of the wild-type. Superimposition of the active-site residues of smPBP5 on PBP5 revealed that perturbation in the orientating key residues may explain the low turnover numbers. Therefore, we establish the involvement of Leu182 in maintaining the physiological and biochemical behaviour of E. coli PBP5.

RcsC is a hybrid histidine kinase that forms part of a phospho-relay signal transduction pathway with RcsD and RcsB. Besides the typical domains of a sensor kinase, i.e. the periplasmic (P), linker (L), dimerization and H-containing (A), and ATP-binding (B) domains, RcsC possesses a receiver domain (D) at the carboxy-terminal domain. To study the role played by each of the RcsC domains, four plasmids containing several of these domains were constructed (PLAB, LAB, AB and ABD) and transformed into Escherichia coli K-12 strain BW25113. Different amounts of biofilm were produced, depending on the RcsC domains expressed: the plasmid expressing the ABD subdomains produced the highest amount of biofilm. This phenotype was also observed when the plasmids were transformed in a ΔrcsCDB strain. Biofilm formation was abolished in the pgaABCD and nhaR backgrounds. The results indicate the existence of a novel signalling pathway that depends on RcsC, yet independent of RcsD and RcsB, that activates the pgaABCD operon and, as a consequence, biofilm formation. This signalling pathway involves the secondary metabolite acetyl phosphate and the response regulator OmpR.

Weissella is a genus of lactic acid bacteria (LAB) consisting of species formerly included in the Leuconostoc paramesenteroides group. Similar to other LAB, they are commonly found in fermented foods but have also been isolated from environmental and human samples. Currently there are 20 recognized species. Herein, three Weissella cibaria genomes were sequenced using Illumia Mi-Seq and Roche 454 technologies. Annotation was performed using the Prokka and JGI IMG pipelines. A thorough analysis of the genomics of the W. cibaria strains was performed, in addition to brief comparative analyses of the genus Weissella as a whole. Genomic sequence data from the newly sequenced W. cibaria strains and data available in GenBank for other Weissella strains was used (n = 10; four Weissella cibaria, one Weissella ceti, one Weissella confusa, one Weissella halotolerans, two Weissella koreensis and one Weissella paramesenteroides). The genomes had sizes varying from 1.3 to 2.4 Mb. DNA G+C contents ranged from 35 to 45 mol%. The core- and pan-proteome at genus and species levels were determined. The genus pan-proteome was found to comprise 4712 proteins. Analysis of the four W. cibaria genomes indicated that the core-proteome, consisting of 729 proteins, constitutes 69 % of the species pan-proteome. This large core-set may explain the divergent niches in which this species has been found. In W. cibaria, in addition to a number of phosphotransferase systems conferring the ability to assimilate plant-associated polysaccharides, an extensive proteolytic system was identified.