INTRODUCTION

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Escherichia coli K-12, the E. coli strain genetically studied in the greatest detail, possesses a single flagellin gene and is therefore monophasic (2, 14). The gene was initially designated hag and recently renamed fliC (10). For a long time, this was believed to describe the phenotypic behavior and genetics of flagellar antigens in E. coli strains in general; the different flagellar antigen serotypes would correspond to different alleles of fliC (hag).

However, some natural isolates of E. coli were then found to contain two alternatively expressed, not cotransducible flagellin genes, hagA and hagB; the former is an allele of the fliC gene of E. coli K-12, while the latter is located at a distance from it (20, 23). It was also shown that the region of hagB is a distinct one integrating in the E. coli K-12 chromosome outside its hitherto known three flagellar regions, as well as far from the site of the integration of the salmonella phase 2 flagellin region. In conformity with the new nomenclature (10), it therefore seems expedient to rename the hagB gene flkA. (The designations of flagellin genes are discussed in the Discussion.) The alteration of the activity of these genes was found to be usually nonreversible (unilateral): fliC^off, flkA^on  fliC^on, flkA^off. The on or off state of fliC correlates with the off or on state of flkA, respectively, because the flk region produces a special (phase-specific) repressor activity similar to that of the product of salmonella repressor gene fljA, acting upon some (sensitive) alleles of fliC; therefore, the existence of a gene similar to fljA that is coexpressed with flkA was postulated (20, 21, 23). New data reported here show an even larger variety in the genetics of flagellar antigens in the natural population of E. coli.

	MATERIALS AND METHODS

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Strains. All of the E. coli strains used are listed in Table 1.

Culture media. Solid, semisolid, and liquid rich media (20) and minimal glucose salt agar (16) were used. Antibacterial drugs, amino acids, nucleic acid bases and/or H antisera were added when appropriate (23).

Crosses. Transductional experiments were carried out by means of temperature-inducible phage P1clr100Km^r-3 (8, 20). Conjugal crosses were carried out in broth (16) for 100 min with Hfr or F' donors containing F' Lac⁺ Km^r (23).

Selection of transductants and exconjugants. Flagellar transductants were selected for motility on semisolid medium supplemented with appropriate anti-H serum to counterselect (by immobilization) bacteria retaining the recipient's flagellar antigen, but no antiserum was used when nonmotile (NM), flagellin-nonproducing strain EJ34 served as the recipient. For the selection of exconjugants by their nutrition requirements, minimal agar with necessary additions was used. H-antigen specificities were determined by means of an agglutination test (20).

Selection of alternative flagellar phase. Three to five transductants of each class were grown on semisolid medium containing antiserum to the expressed H antigen, thus providing strict conditions for testing of their ability to produce an alternative flagellar antigen phase (20, 22).

	RESULTS

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Identification of a new flagellin locus in the standard test strains for H55 (E2987-73) and H44 (781-55). It was easy to propagate the phage used (P1clr100Km^r-3) on strain E2987-73 but not on strain 781-55. Therefore, fertile bacteria produced from the latter by introduction of plasmid F' Lac⁺ Km^r were mated with strain PM298, selecting for Str^r Rif^r His⁺ recombinants; one that was motile on semisolid agar containing antiserum H48 and exhibited flagellar antigen H44 was designated PM304 (Table 1) and used for following genetic analysis.

Cryptic flagellin genes in standard strains 781-55 and E2987-73. When transductions similar to those in experiment A of Table 2 were carried out with donors PM304 (H44) and E2987-73 (H55) and recipient B99-2 (whose H6 antigen is determined by fliC₆, which is insensitive to phase-specific repression), the results were different: all of the transductants obtained were either H4 (with the donor PM304) or H38 (with the donor E2987-73), and no transductants were found with H44 or H55 (Table 2, experiment D). This indicated, first of all, that the fliC allele, fliC₆, of the recipient B99-2 was not repressed by fllA₄₄ or fllA₅₅ and, furthermore, that the genes encoding flagellar antigens H4 and H38 were present (but not expressed) in the donor strains and then expressed in transductants. Why were they not expressed in the donor strains? Were they under phase-specific repression controlled by the fllA region?

Identification of another new flagellin region in E223-69, the standard test strain for flagellar antigen H54. H54 transductants were obtained with E223-69 as the donor and strain EJ34 (NM) or PM336 (H16) as the recipient, but attempts to obtain similar transductants from four other recipient strains (PM335 with H48, PM315 with H3, PM304 with H44, and PM471 with H55) failed (Table 4, experiment A), despite the close relationship of three of them to EJ34 or PM336 (PM335 was the immediate ancestor of PM336, and PM315 and PM471 were derived directly from EJ34). The most probable interpretation is that a gene responsible for H54 can be inherited and expressed in strains of the E. coli K-12 line (EJ34 and PM336) but is not able to replace or suppress any of the flagellin genes fliC₄₈, flkA₃, fllA₄₄, and fllA₅₅. On the other hand, the tested H54 transductants arising from recipient strain PM336 (H16) were able to produce an alternative flagellar phase, H16, thus indicating that the expression of allele fliC₁₆ (which is known to be sensitive to phase-specific repression) was governed by the introduced flagellin gene region of the donor. In this respect, the latter resembled the flkA₃ region of E. coli and the salmonella phase 2 flagellin region, and therefore it seemed interesting to look into its interaction with the relevant salmonella flagellin gene. So, transduction was carried out with donor strain EJ262 containing the salmonella phase 2 flagellin region in an active state and recipient strain PM472 (an H54-expressing transductant of EJ34) (Table 4, experiment B). No motile transductants were obtained on semisolid medium containing anti-H54 serum, indicating that, most probably, the gene for H54 could not be replaced with salmonella phase 2 flagellin gene fljB; the control transduction with recipient strain PM336 (H16) yielded the expected transductants expressing salmonella flagellar antigen e,n,x and capable of phase variation between it and H16.

	DISCUSSION

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The experimental data presented here show an unexpected diversity of the flagellin gene pool in the E. coli population in nature. These findings, as well as other published data on the variety of E. coli flagellar antigen genetics, are summarized in Table 6. In this investigation, two new loci, fllA (alleles fllA₄₄ and fllA₅₅, determining antigens H44 and H55 in E. coli standard H-test strains 781-55 and E2987-73, respectively) and flmA (allele flmA₅₄ for antigen H54 in E. coli standard H-test strain E223-69), were discovered and shown, when being transferred in E. coli K-12 derivatives, each to have a different chromosomal localization than any of the previously identified flagellin genes of E. coli: fllA was closely linked to pheA2 (map position 57) and showed no linkage to mtlA2 (map position 81), while flmA was closely linked to the latter; fliC, the single flagellin gene of E. coli K-12 (10), is located at position 43 (2), and flkA (formerly hagB) is very close to argG6 (map position 69) (23). flkA (23), fllA, and flmA are not alleles of salmonella gene fljB because it does not replace them. The insertion site of the latter into the E. coli K-12 chromosome is at map position ca. 55 (6). The results of the conjugational crosses confirmed the proposals and conclusions inferred from the experiments carried out by means of transduction that fliC, flkA, fllA, flmA, and salmonella fljB belong to distinct flagellar regions and therefore, when transduced, are unable to replace one another.

As to the nature of the integration into the E. coli K-12 chromosome of the flagellin gene regions other than that of fliC, one cannot exclude the possible presence in the relevant sites of the E. coli K-12 chromosome of sequences displaying at least some homology to these flagellar regions (flagellin genes), although the integration might be determined by sequences flanking rather a big P1-transduced piece of the chromosome.

The two newly described flagellar regions differ in the ability to suppress alleles of fliC sensitive to phase-specific repression. That of flmA₅₄ possesses this activity and is thus similar in this respect to the salmonella phase 2 flagellin region and the E. coli flk region. In contrast, the fll region (for both flagellin gene alleles fllA₄₄ and fllA₅₅) lacks such a feature, and particularly in this connection one may expect the existence of E. coli strains that express two flagellin genes. Indeed, I found naturally occurring E. coli strains of serogroup O18 exhibiting two different flagellar antigens, H14 and H55, simultaneously (24).

Concerning the gene nomenclature used, it is worth noting again that of the above-named E. coli flagellin genes, only one (fliC) has been found in E. coli K-12 and that this strain represents a genetically rather rare clone among the naturally occurring E. coli strains (5). Nevertheless, the principle developed for the new unified nomenclature of the E. coli K-12 and Salmonella typhimurium flagellar genes (10) seems to be also applicable for designation of the E. coli flagellar genes not reported to occur in these strains. The principle is based on the fact that the flagellar genes gather in three (E. coli K-12) or four (S. typhimurium) separate chromosome regions (14). Each region is assigned a three-letter symbol starting with fl, while the third letter is determined by the relevant flagellar region and used in alphabetical order starting with g. Therefore, three similar regions in E. coli K-12 and S. typhimurium have been designated flg, flh, and fli and the phase 2 flagellin region of S. typhimurium has been designated flj. Inside a region, the genes (to be distinguished) are given alphabetical (capital letter) extensions according to their genome order (e.g., fljA and fljB); however, exceptions to the latter rule are allowed (10). According to this principle, the designations flk, fll, and flm were used for the three distinct flagellin regions not found in E. coli K-12, with the extension A for the flagellin-specifying genes, and a subsequent subscript extension for an allele indicating the serotype of the flagellin determined by it.

However, sometimes the use of the principle seems not to be so simple regarding wild strains. Some wild strains may possess several (at least two) flagellin genes in the same region. For example, in E. coli P12b, the standard test strain for E. coli antigen H17, two flagellin genes formerly designated hagA₄ (assumed for an allele of hag, i.e., fliC) and hagC₁₇ were reported, and owing to their high cotransducibility (22), both should be considered constituents of the same third flagellar region (fli). The difficulty of naming such flagellin genes arises from the absence of any direct criteria for identifying genuine fliC in strain P12b, as well as in other wild E. coli strains, especially in view of the fact that the genome arrangement, at least in the vicinity near the flagellin gene(s) in the fli region in some wild strains, may be different from that of E. coli K-12. Therefore, the designations fliC' and fliC" seem to be useful with respect to P12b; on the other hand, if only a single flagellin gene is found in this flagellar region, the gene designation fliC without any specification seems the most appropriate.

Phase-specific repressor sensitivity or resistance determined by salmonella gene fljA or by its analog (e.g., flkB, according to the principle of the new nomenclature) postulated in E. coli (20, 23) is not an intrinsic feature of fliC, as fliC₄₈ of E. coli K-12 is resistant, while fliC_i of S. typhimurium is sensitive. Besides, other sensitive (hagA₂, hagA₁₆, and hagA₂₁) and resistant (hagA₄ and hagA₆) alleles of fliC have been reported in wild E. coli strains (15, 20-23). On the other hand, genes insensitive to such kinds of repression may be cryptic for a different yet enigmatic reason, as was reported for cryptic gene fliC'₄ of strain P12b, which became active when fliC"₁₇ was spontaneously suppressed (22). Therefore, the presence of cryptic genes fliC₄ and fliC₃₈ in strains 781-55 (H44) and E2987-73 (H55), respectively, seems to be not extraordinary. Interestingly, however, the attempts to obtain these alleles in an active state by transducing them into E. coli K-12 derivatives failed (Table 2, experiment A), but it turned out to be possible when strain B99-2 (H6) was used as the recipient (Table 2, experiment D). The inability of H4 and H38 transductants to restore the H6 phenotype indicated that fliC₆ was not repressed but replaced by a donor flagellin gene or a sequence closely linked to and inserted into the recipient chromosome together with it. Therefore, the genes encoding H4 and H38 should be considered constituents of the third flagellar region and designated fliC₄ and fliC₃₈, respectively. It may be that the discrepancy between the results of transduction to different recipients depended on the probable difference between the genome arrangements in the vicinities of fliC₄₈ (E. coli K-12) and fliC₆ (B99-2) and that the silent state of fliC₄ and fliC₃₈ resulted from a very closely linked alteration.

The methods of transduction and conjugation used in this study were fruitful in discovering new phenomena and provided cogent arguments for the above-stated conclusions which gave new knowledge about E. coli flagellar genetics concerning the existence of the new flagellar regions and flagellin genes, as well as the new examples corroborating the natural occurrence of E. coli strains each containing two flagellin genes. Molecular genetic analysis should be used for further investigation of the phenomena, but it is inaccessible to me because of the catastrophic situation of science in my country.

The genetics of bacterial flagellins has recently become more complicated in other taxonomic groups, too (3, 4, 9, 11, 12, 17, 25-27). Although in E. coli the genetics of H-antigen specificity, like that of other features, was earlier focused on K-12, there have been rare attempts to study it in other E. coli wild strains (15, 18). The data presented here, together with those reported earlier (20-24), show that the knowledge of the flagellar genetics of E. coli K-12 only partially reflects the much more complicated and rather diverse flagellar genetics of the bacterial population covered by the taxonomic term E. coli.