Hetero-oligomeric cell wall channels (porins) of Nocardia farcinica

Abstract

The cell wall of Nocardia farcinica contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of N. farcinica for negatively charged antibiotics. Based on partial sequencing of the protein responsible for channel formation derived from N. farcinica ATTC 3318 we were able to identify the corresponding genes (nfa15890 and nfa15900) within the known genome of N. farcinica IFM 10152. The corresponding genes of N. farcinica ATTC 3318 were separately expressed in the Escherichia coli BL21DE3Omp8 strain and the N-terminal His10-tagged proteins were purified to homogeneity using immobilized metal affinity chromatography. The pure proteins were designated NfpANHis and NfpBNHis, for N. farcinica porin A and N. farcinica porin B. The two proteins were checked separately for channel formation in lipid bilayers. Our results clearly indicate that the proteins NfpANHis and NfpBNHis expressed in E. coli could only together form a channel in lipid bilayer membranes. This means that the cell wall channel of N. farcinica is formed by a heterooligomer. NfpA and NfpB form together a channel that may structurally be related to MspA of Mycobacterium smegmatis based on amino acid comparison and renaturation procedure.

Functional Expression of the PorAH Channel from Corynebacterium glutamicum in Cell-free Expression Systems

“…They show in addition that the PorAPorH channel shares the characteristic fingerprints of channels such as bacterial porin (34) and also mitochondrial porin (35): closure at positive and negative potential with fast and slow kinetics, asymmetric voltage dependence, and multiple conductance states. Similar porin channels can be found in related families of bacteria, C. diphtheria, C. efficiens, and N. farcinia (8,36,37).…”

Discovery of a cell wall porin in the mycolic‐acid‐containing actinomycete Dietzia marisDSM 43672

“…In this study, we identified a channel from the cell wall of D. maris DSM 43672. A protein responsible for channel formation was identified with a molecular mass of about 120 kDa, which may be formed from a homooligomer of smaller polypeptides similar to the situation in other members of mycolata, where the cell wall channels are frequently formed by oligomers [18][19][20][21]. According to results of the lipid bilayer conductance measurements, the cell wall channel of D. maris DSM 43672 is wide, water filled and has a high preference for the passage of cations.…”

“…However, some exhibit cation selectivity (as in D. maris) whereas others are anion-selective, although it is likely that each organism will possess channels with both types of selectivity in order to facilitate uptake of the full range of hydrophilic solutes. More interestingly, some porins appear to work as hetero-oligomers [18,46] whereas others form likely homo-oligomers [47].…”

“…This means that the mycolic acid layer has the same function as the outer membranes of Gram-negative bacteria, which contain porins for the passage of hydrophilic solutes. Accordingly, channel-forming proteins with characteristics similar to the porins of their Gram-negative counterparts have been identified as existing in the cell walls of multiple members of the mycolata, including Mycobacterium chelonae and Mycobacterium bovis [15,16], Nocardia farcinica [17,18], Corynebacterium diphtheriae, Corynebacterium glutamicum, Corynebacterium amycolatum [19][20][21][22][23], Rhodococcus equi and Rhodococcus erythropolis [24,25]. It is therefore likely that channel-forming proteins are widely distributed in the mycolata and are responsible for the uptake of hydrophilic compounds across the mycolic acid layer.…”

Identification and characterization of a cell wall porin from <i>Gordonia jacobaea</i>

“…Some of these channel-forming proteins are related to the two major families MspA and PorA (Faller et al, 2004). The MspA family is present in the genera Mycobacterium (Hillmann et al, 2007), Rhodococcus (Somalinga and Mohn, 2013), Nocardia (Kläckta et al, 2011) and Tsukamurella (Dörner et al, 2004). PorA-like proteins have exclusively been found in the genus Corynebacterium (Costa-Riu et al, 2003).…”