Isolation and characterization of insertional mutations in flagellin genes in the archaeon Methanococcus voltae

Abstract

Methanococcus voltae is a flagellated member of the Domain Archaea that has four flagellin genes arranged in two transcriptional units. One transcriptional unit encodes only flaA while the second is a multi-cistronic unit encoding three flagellin genes (flaB1, flaB2, and flaB3) as well as at least seven other open reading frames downstream. The polymerase chain reaction was used to amplify an internal fragment of the flaA gene which was subsequently cloned into an insertion vector developed for M. voltae. Transformation of protoplasts with this vector led to the isolation of mutant strains that had insertions in flaA or flaB2. Mutant strains carrying insertions in flaA had flagelia that were similar to wild-type cells in both number and appearance when viewed using the electron microscope. In addition, some of these mutant strains had profiles identical to the wild type in immunoblots developed with antisera raised against the 31 kDa flagellin of M. voltae. All flaA mutant strains and the wild-type cells showed immuno-cross-reactive bands at 33 and 31 kDa (corresponding to purified flagellins) as well as at 18 kDa. Some flaA mutant strains also showed an immuno-cross-reactive band at 27 kDa which probably represents a truncated flagellin produced by the insertion vector. However, both types of flaA mutant strains were less motile than the wild type in semi-swarm plate experiments. The mutant strain with an insertion in flaB2 was non-flagellated when examined by electron microscopy and it was non-motile in semi-swarm plate experiments. It represents the first structural mutant strain isolated in a methanogen. This mutant strain lacked the 33, 31, and 18 kDa immuno-cross-reactive bands observed in the wild type and flaA mutant strains, and instead had a novel band at 20 kDa. This band may represent an unmodified flagellin which still has an attached leader peptide. If so, then one of the downstream genes in the multi-cistronic transcriptional unit may encode a leader peptidase for the flagellin system.

Archaeal genetics — the third way

“…In this approach, recombination between a truncated version of the gene and its chromosomal copy leads to an insertion/disruption mutation ( Figure 2B). A targeted version of insertional mutagenesis has been used in M. voltae, to characterise genes encoding flagellins and hydrogenases [95][96][97] . For random mutagenesis, a genomic library of small fragments (less than a full-length gene) is used to target recombination.…”

In vivo transposon mutagenesis of the methanogenic archaeon Methanosarcina acetivorans C2A using a modified version of the insect mariner -family transposable element Himar1

“…Most genetic studies of methanoarchaea published to date have involved directed mutagenesis of previously identified genes using gene replacement methods (4,(11)(12)(13). Although such studies are invaluable for examining the roles of specific genes in specific functions, they are of limited use in identifying new roles for these genes, because one must have a specific trait in mind when testing the phenotypic consequences of mutations in a given gene.…”

Posttranslational Processing ofMethanococcus voltaePreflagellin by Preflagellin Peptidases ofM. voltaeand Other Methanogens

“…One transcriptional unit contains only flaA; the other, polycistronic transcriptional unit (at least 5.4 kb in length), containing flaB1, flaB2, flaB3, and a number of presumed flagellum accessory genes, initiates at flaB1 and extends to at least the end of flaG (13; D. P. Bayley, J. D. Correia, and K. F. Jarrell, unpublished data). N-terminal (14), transcriptional (13), and mutational (9) analyses have provided evidence that flaB1 and flaB2 encode the major flagellins in M. voltae. Comparison of the N-terminal sequence of one purified flagellin with the amino acid sequence predicted from the gene sequence (13) confirmed the presence of a 12-amino-acid leader peptide.…”

“…A nonflagellated M. voltae mutant (M. voltae P-2 [9]) was also examined for preflagellin peptidase activity under the standard reaction conditions. This mutant has an insertional vector located in flaB2, which also results in a polar effect on the cotranscribed downstream genes.…”