Cell-bound exopolysaccharides from an axenic culture of the intertidal mudflat Navicula phyllepta diatom affect biofilm formation by benthic bacteria

International audienceAt low tide, intertidal mudflat biofilms cover large surfaces and are mainly responsible for the high productivity of these marine areas. In the European Atlantic coast, such biofilms are mainly composed of diatoms, especially Navicula phyllepta, bacteria, and microbial extracellular polymeric substances (EPS). To better understand interactions occurring between microorganisms, we first axenized a N. phyllepta culture with a new and simple protocol. Colloidal and bound EPS secreted by diatom cells during the exponential growth and the stationary phase were then harvested, and we tested their effects on the in vitro formation of biofilms by three marine bacteria. The latter had been isolated from a French Atlantic intertidal mudflat and were previously selected for their strong in vitro biofilm-forming ability. They belong to the Flavobacterium, Roseobacter, and Shewanella genera. Navicula phyllepta-bound EPS synthesized during the stationary phase specifically inhibited the biofilm formation by the Flavobacterium sp. strain, whereas they stimulated biofilm development by the two other strains. The EPS acted in all cases during the first stages of the biofilm establishment. Saccharidic molecules were found to be responsible for these activities. This is the first report on marine bacterial antibiofilm saccharides of microalgal origin. This work points out the complexity of the benthic natural biofilms with specific microalgae/bacteria interactions and underlines the possibility to use axenic diatoms as a source of bioactive compounds

On the bacterial communities associated with the corrosion product layer during the early stages of marine corrosion of carbon steel

International audienc

Mechanism of Bactericidal Activity of Microcin L in Escherichia coli and Salmonella enterica▿

For the first time, the mechanism of action of microcin L (MccL) was investigated in live bacteria. MccL is a gene-encoded peptide produced by Escherichia coli LR05 that exhibits a strong antibacterial activity against related Enterobacteriaceae, including Salmonella enterica serovars Typhimurium and Enteritidis. We first subcloned the MccL genetic system to remove the sequences not involved in MccL production. We then optimized the MccL purification procedure to obtain large amounts of purified microcin to investigate its antimicrobial and membrane properties. We showed that MccL did not induce outer membrane permeabilization, which indicated that MccL did not use this way to kill the sensitive cell or to enter into it. Using a set of E. coli and Salmonella enterica mutants lacking iron-siderophore receptors, we demonstrated that the MccL uptake required the outer membrane receptor Cir. Moreover, the MccL bactericidal activity was shown to depend on the TonB protein that transduces the proton-motive force of the cytoplasmic membrane to transport iron-siderophore complexes across the outer membrane. Using carbonyl cyanide 3-chlorophenylhydrazone, which is known to fully dissipate the proton-motive force, we proved that the proton-motive force was required for the bactericidal activity of MccL on E. coli. In addition, we showed that a primary target of MccL could be the cytoplasmic membrane: a high level of MccL disrupted the inner membrane potential of E. coli cells. However, no permeabilization of the membrane was detected

Biofilms in a marine environment: example of intertidal mudflats and metallic port structures

International audienc

Genetic Analysis and Complete Primary Structure of Microcin L

Escherichia coli LR05, in addition to producing MccB17, J25, and D93, secretes microcin L, a newly discovered microcin that exhibits strong antibacterial activity against related Enterobacteriaceae, including Salmonella enterica serovars Typhimurium and Enteritidis. Microcin L was purified using a two-step procedure including solid-phase extraction and reverse-phase C(18) high-performance liquid chromatography. A 4,901-bp region of the DNA plasmid of E. coli LR05 was sequenced revealing that the microcin L cluster consists of four genes, mclC, mclI, mclA, and mclB. The structural gene mclC encoded a 105-amino-acid precursor with a 15-amino-acid N-terminal extension ending with a Gly-Ala motif upstream of the cleavage site. This motif is typical of the class II microcins and other gram-positive bacteriocins exported by ABC transporters. The mclI immunity gene was identified upstream of the mclC gene and encodes a 51-amino-acid protein with two potential transmembrane domains. Located on the reverse strand, two genes, mclA and mclB, encoded the proteins MclA and MclB, respectively. They bear strong relatedness with the ABC transporter proteins and accessory factors involved in the secretion of microcins H47, V, E492, and 24. The microcin L genetic system resembles the genetic organization of MccV. Furthermore the MccL primary structure has been determined. It is a 90-amino-acid peptide of 8,884 Da with two disulfide bridges. The N-terminal region has significant homologies with several gram-positive bacteriocins. The C-terminal 32-amino-acid sequence is 87.5% identical to that of MccV. Together, these results strongly indicate that microcin L is a gram-negative class II microcin