Identification of a cation-specific channel (TipA) in the cell wall of the gram-positive mycolata Tsukamurella inchonensis: the gene of the channel-forming protein is identical to mspA of Mycobacterium smegmatis and mppA of Mycobacterium phlei

Abstract

Detergent extracts of whole cells of the Gram-positive bacterium Tsukamurella inchonensis ATCC 700082, which belongs to the mycolata, were studied for the presence of ion-permeable channels using lipid bilayer experiments. One channel with a conductance of about 4.5 nS in 1 M KCl was identified in the extracts. The channel-forming protein was purified to homogeneity by preparative SDS-PAGE. The protein responsible for channel-forming activity had an apparent molecular mass of about 33 kDa as judged by SDS-PAGE. Interestingly, the protein showed cross-reactivity with polyclonal antibodies raised against a polypeptide derived from MspA of Mycobacterium smegmatis similarly as the cell wall channel of Mycobacterium phlei. Primers derived from mspA were used to clone and sequence the gene of the cell wall channels of T. inchonensis (named tipA for T. inchonensis porin A) and M. phlei (named mppA for M. phlei porin A). Surprisingly, both genes, tipA and mppA, were found to be identical to mspA of M. smegmatis, indicating that the genomes of T. inchonensis, M. phlei and M. smegmatis contain the same genes for the major cell wall channel. RT-PCR revealed that tipA is transcribed in T. inchonensis and mppA in M. phlei. The results suggest that despite a certain distance between the three organisms, their genomes contain the same gene coding for the major cell wall channel, with a molecular mass of 22 kDa for the monomer.

Corynebacterium jeikeium jk0268 Constitutes for the 40 Amino Acid Long PorACj, Which Forms a Homooligomeric and Anion-Selective Cell Wall Channel

“…The many similarities between PorA/PorH and PorACj suggest that these channel-forming proteins form a family of proteins analogous to the MspABCD cell wall channel family of M. smegmatis and related species [58], [73]–[75]. Even though PorACj structurally differs from the cell wall channel composition of the heterooligomeric channels within in the genus Corynebacterium there is a clear evidence for phylogenic relationships of the investigated species.…”

Draft Genome Sequence of the Bacterium Gordonia jacobaea , a New Member of the Gordonia Genus

“…In the last decades several investigators have pointed out the existence of proteins playing a similar role to that of the porins of Gram-negative bacteria, facilitating the diffusion of hydrophilic solutes through the bacterial envelopes and the entry of antibiotics ( 6 , 7 ). The first description of these porins occurred in the 90s, when a porin of Mycobacterium chelonae was characterized ( 8 ); since then, other porins in genera like Mycobacterium ( 9 , 10 ), Tsukamurella ( 11 ), or Nocardia ( 12 ) have been reported.…”

Cell wall channels of Rhodococcus species: identification and characterization of the cell wall channels of Rhodococcus corynebacteroides and Rhodococcus ruber

“…They contain mostly α-helical structures in the thin mycolic acid layer of the Corynebacteria (Ziegler et al 2008 ; Barth et al 2010 ; Abdali et al 2013 ). Pores in the thicker mycolic acid layer of Mycobacteria , Nocardia and Tsukamorella are all formed from oligomers (octamers) of polypeptides with a molecular mass around 20 kDa that are identical or similar in sequence or mass to the subunit MspA of the Mycobacterium smegmatis cell wall channel (Niederweis et al 1999 ; Riess et al 2001 ; Dörner et al 2004 ; Kläckta et al 2011 ). Octamers of MspA form a goblet-like structure with one channel where the MspA monomer contributes two beta strands to the octameric beta barrel with nonpolar outer surfaces around the pore (Faller et al 2004 ).…”