Agrobacterium tumefaciens twin-arginine-dependent translocation is important for virulence,flagellation, and chemotaxis but not type IV secretion

This study characterized the contribution of the twin-arginine translocation (TAT) pathway to growth, motility, and virulence of the phytopathogen Agrobacterium tumefaciens. In contrast to wild-type strain A348, a tatC null mutant failed to export the green fluorescent protein fused to the trimethylamine N-oxide reductase (TorA) signal sequence or to grow on nitrate as a sole electron acceptor during anaerobic growth. The tatC mutant displayed defects in growth rate and cell division but not in cell viability, and it also released abundant levels of several proteins into the culture supernatant when grown in rich medium or in vir induction minimal medium. Nearly all A348 cells were highly motile in both rich and minimal media. By contrast, approximately 0.1% of the tatC mutant cells were motile in rich medium, and <0.01% were motile in vir induction medium. Nonmotile tatC mutant cells lacked detectable flagella, whereas motile tatC mutant cells collected from the edge of a motility halo possessed flagella but not because of reversion to a functional TAT system. Motile tatC cells failed to exhibit chemotaxis toward sugars under aerobic conditions or towards nitrate under anaerobic conditions. The tatC mutant was highly attenuated for virulence, only occasionally (approximately 15% of inoculations) inciting formation of small tumors on plants after a prolonged incubation period of 6 to 8 weeks. However, an enriched subpopulation of motile tatC mutants exhibited enhanced virulence compared to the nonmotile variants. Finally, the tatC mutant transferred T-DNA and protein effectors to plant cells and a mobilizable IncQ plasmid to agrobacterial recipients at wild-type levels. Together, our findings establish that, in addition to its role in secretion of folded cofactor-bound enzymes functioning in alternative respiration, the TAT system of A. tumefaciens is an important virulence determinant. Furthermore, this secretion pathway contributes to flagellar biogenesis and chemotactic responses but not to sensory perception of plant signals or the assembly of a type IV secretion system.     

Stalkless mutants of Caulobacter crescentus.

A stalk, a single falgellum, several pili, and deoxyribonucleic acid (DNA) phage receptors are polar surface structures expressed at a defined time in the Caulobacter crescentus cell cycle. When mutants were isolated as DNA phage phiCbK-resistant or ribonucleic acid (RNA) phage phiCp2-resistant, as well as nonmotile, strains, 5 out of 30 such mutant isolates were found not to possess stalks, but did possess inactive flagella. These stalkless mutants were resistant simultaneously to both DNA and RNA phages and did not possess pili and DNA pendent stalkless mutants. All motile revertants simultaneously regained the capacity to form stalks and susceptibility to DNA and RNA phages. It is suggested that a single mutation pleiotropically affects stalk formation, flagella motility, and coordinate polar morphogenesis of pili and DNA phage receptors. The stalkless mutants grew at a generation time similar to that of the wild-type strain at 30 degrees C. Cell size and morphology of a stalkless mutant, C. crescentus CB13 pdr-819, were also similar to those of the wild-type strain, except for the absence of a stalk. In addition, the CB13 pdr-819 predivisional cells were partitioned into smaller and larger portions, indicating asymmetrical cell division, as in the wild-type strain. From these results, it is suggested that swarmer cells undergo transition to cells of a stalked-cell nature without stalk formation and that the cell cycle of the stalkless mutant proceeds in an ordered sequence similar to that defining the wild-type cell cycle.     

Non-motile mini-transposon mutants of Bordetella bronchiseptica exhibit altered abilities to invade and survive in eukaryotic cells

Non-motile mutants of Bordetella bronchiseptica were generated after mini-transposon mutagenesis. One non-motile mutant (designated VMM1) was derived from the bvg-positive strain BB7865 and four mutants (designated AMM1–4) were derived from the isogenic bvg-negative strain BB7866. Southern hybridisation analysis indicated that loss of motility was not due to the disruption of the flagellin subunit gene. Western blot and transmission electron microscopic analysis indicated that three of the five mutants expressed neither the flagellin subunit (40 kDa) nor flagella whereas one mutant expressed intact flagella under all conditions tested. One unique bvg-negative mutant, AMM4, exhibited temperature-dependent repression of flagella biosynthesis and motility at 37°C. The ability of AMM4 to invade and survive in HeLa cells was significantly decreased.     

Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri.

Vibrio fischeri is found both as a free-living bacterium in seawater and as the specific, mutualistic light organ symbiont of several fish and squid species. To identify those characteristics of symbiosis-competent strains that are required for successful colonization of the nascent light organ of juvenile Euprymna scolopes squids, we generated a mutant pool by using the transposon Mu dI 1681 and screened this pool for strains that were no longer motile. Eighteen independently isolated nonmotile mutants that were either flagellated or nonflagellated were obtained. In contrast to the parent strain, none of these nonmotile mutants was able to colonize the juvenile squid light organ. The flagellated nonmotile mutant strain NM200 possessed a bundle of sheathed polar flagella indistinguishable from that of the wild-type strain, indicating that the presence of flagella alone is not sufficient for colonization and that it is motility itself that is required for successful light organ colonization. This study identifies motility as the first required symbiotic phenotype of V. fischeri.     

Temperature and pyoverdine-mediated iron acquisition control surface motility of Pseudomonas putida

Pseudomonas putida KT2440 is unable to swarm at its common temperature of growth in the laboratory (30 degrees C) but exhibits surface motility similar to swarming patterns in other Pseudomonas between 18 degrees C and 28 degrees C. These motile cells show differentiation, consisting on elongation and the presence of surface appendages. Analysis of a collection of mutants to define the molecular determinants of this type of surface movement in KT2440 shows that while type IV pili and lipopolysaccharide O-antigen are requisites flagella are not. Although surface motility of flagellar mutants was macroscopically undistinguishable from that of the wild type, microscopy analysis revealed that these mutants move using a distinct mechanism to that of the wild-type strain. Mutants either in the siderophore pyoverdine (ppsD) or in the FpvA siderophore receptor were also unable to spread on surfaces. Motility in the ppsD strain was totally restored with pyoverdine and partially with the wild-type ppsD allele. Phenotype of the fpvA strain was not complemented by this siderophore. We discuss that iron influences surface motility and that it can be an environmental cue for swarming-like movement in P. putida. This study constitutes the first report assigning an important role to pyoverdine iron acquisition in en masse bacterial surface movement.     

Symbiotic and competitive properties of motility mutants of Rhizobium trifolii TA1

Non-motile mutants of Rhizobium trifolii defective in either flagellar synthesis or function were isolated by transposon Tn5 mutagenesis. they were indistinguishable from motile control strains in growth in both laboratory media and in the rhizosphere of clover roots. When each non-motile mutant was grown together with a motile strain in continuous culture, the numbers of motile and non-motile organisms remained in constant proportion, implying that their growth rates were essentially identical. When inoculated separately onto clover roots, the mutants and wildtype did not differ significantly in the number of nodules produced or in nitrogen fixing activity. However, when mixtures of equal numbers of mutant and wild-type cells were inoculated onto clover roots, the motile strain formed approximately five times more nodules than the flagellate or non-flagellate, non-motile mutants, suggesting that motility is a factor in competition for nodule formation.     

Possible involvement of bacterial autolytic enzymes in flagellar morphogenesis.

Autolytic enzymes were found to be required for flagellar morphogenesis in Bacillus subtilis 168 and Bacillus licheniformis 6346. Two previously characterized, poorly lytic, chain-forming mutants of B. subtilis 168, strains FJ3 (temperature conditional) and FJ6, each 90 to 95% deficient in the production of N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase, were observed to be nonmotile at 35 degrees C in a variety of liquid and semisolid meida. In contrast, cells of the isogenic wild-type strain were motile and fully separated. Electron microscopy revealed the complete absence of flagella on the mutant cells. Similar observations were made with another poorly lytic strain of B. subtilis 168 (Nil5) and with two poorly lytic, phosphoglucomutase-deficient mutants of B. licheniformis 6346 (MH-3, MH-5). In minimal media lacking galactose (restrictive conditions), the B. licheniformis mutants failed to form flagella, or had serious abnormalities in flagellar morphogenesis and motility. Under permissive conditions, mutants FJ3 (grown at 17 degrees C) and MH-5 (grown with addend galactose) showed increased autolytic activities, grew in the dechained form, and regained their capacities to synthesize functional flagella. Examination of several classes of spontaneous revertants derived from the various mutant strains further demonstrated a close relationship between autolysin acttivity and flagellation in the two Bacillus spp.     

Slow motile mutant in Salmonella typhimurium

Enomoto, Masatoshi (National Institute of Genetics, Misima, Japan). Slow motile mutant in Salmonella typhimurium. J. Bacteriol. 90:1696-1702. 1965.-A slow motile mutant, SJ399, was isolated from a wild-type strain of Salmonella typhimurium TM2. The mutant was as motile as the wild type in broth culture at 37 C. However, on semisolid medium it produced a much narrower swarming band than TM2. The motility of this mutant was hindered by the viscosity of semisolid medium. H antigenicity and morphological characters of flagella of the mutant were the same as those of the wild type. The motility phage, chi, responded differently to SJ399 and the wild type. Plaques of SJ399 were small and cloudy, whereas on the wild type they were large and clear. The efficiency of plating on SJ399 was 0.36 as compared with 1 with the wild type. Stained preparations revealed that the mutant had about one-third the number of flagella of the wild type. The reduction of the number of flagella also was ascertained by biochemical measurement of flagellar protein which was purified after deflagellation from cells. The content of flagellin in SJ399 was about 32% of that of the wild type. Phage P22-mediated transductions from SJ399 to nonflagellated (fla(-)) and paralyzed (mot(-)) mutants showed that the mutant SJ399 complements seven fla(-) and three mot(-) strains which are representative mutants of flagellation and motility cistrons, respectively. The mutation site of SJ399 was cotransduced with both motA and B cistrons. The two point cross tests between SJ399 and mot mutants revealed that the mutation site of SJ399 is located in the motB cistron. The insertion of the genetic region containing the mutation site of SJ399 to the motB cistron is discussed in relation to intracistronic complementation.     

Vibrio fischeri flagellin A is essential for normal motility and for symbiotic competence during initial squid light organ colonization

The motile bacterium Vibrio fischeri is the specific bacterial symbiont of the Hawaiian squid Euprymna scolopes. Because motility is essential for initiating colonization, we have begun to identify stage-specific motility requirements by creating flagellar mutants that have symbiotic defects. V. fischeri has six flagellin genes that are uniquely arranged in two chromosomal loci, flaABCDE and flaF. With the exception of the flaA product, the predicted gene products are more similar to each other than to flagellins of other Vibrio species. Immunoblot analysis indicated that only five of the six predicted proteins were present in purified flagella, suggesting that one protein, FlaF, is unique with respect to either its regulation or its function. We created mutations in two genes, flaA and flaC. Compared to a flaC mutant, which has wild-type flagellation, a strain having a mutation in the flaA gene has fewer flagella per cell and exhibits a 60% decrease in its rate of migration in soft agar. During induction of light organ symbiosis, colonization by the flaA mutant is impaired, and this mutant is severely outcompeted when it is presented to the animal as a mixed inoculum with the wild-type strain. Furthermore, flaA mutant cells are preferentially expelled from the animal, suggesting either that FlaA plays a role in adhesion or that normal motility is an advantage for retention within the host. Taken together, these results show that the flagellum of V. fischeri is a complex structure consisting of multiple flagellin subunits, including FlaA, which is essential both for normal flagellation and for motility, as well as for effective symbiotic colonization.     

Nonsense Motility Mutants in SALMONELLA TYPHIMURIUM

Of 313 motility-deficient mutants isolated from an LT2 his(amber) strain fixed in phase 1 by gene vh2(-), 25 regained motility when amber or ochre suppressors were introduced, in F' factors or by transduction. The fla mutants (23 amber, 1 ochre) fell in complementation groups A, B, C, F, K, a new group, M, and at least one further new group; the hypothesis of a fla gene which specifies only an RNA structural component of a flagellum-synthesizing basal apparatus is disproven for the corresponding genes. Hfr and transductional crosses confirmed gene assignments from complementation and indicated that flaM and another new fla locus map near H1. A small minority of motile bacteria were detectable in many of the amber fla mutants. In groups A and F some pairs of amber fla mutants complemented each other, and perhaps each of these groups corresponds to more than one structural gene. The suppressed derivatives of a mutant with an amber mutation in H1 made flagella morphologically and serologically indistinguishable from wild-type flagella. A slow-spreading but flagellate mutant showed mainly non-translational motility in broth, and in a viscous medium the bacteria reversed very frequently; its amber mutation, probably near H1, is inferred to cause a defect in chemotaxis, so that the bacteria give the avoidance reaction continuously.     

Analysis of the role of the two flagella of Bradyrhizobium japonicum in competition for nodulation of soybean

Bradyrhizobium japonicum has two types of flagella. One has thin filaments consisting of the 33-kDa flagellins FliCI and FliCII (FliCI-II) and the other has thick filaments consisting of the 65-kDa flagellins FliC1, FliC2, FliC3, and FliC4 (FliC1-4). To investigate the roles of each flagellum in competition for nodulation, we obtained mutants deleted in fliCI-II and/or fliC1-4 in the genomic backgrounds of two derivatives from the reference strain USDA 110: the streptomycin-resistant derivative LP 3004 and its more motile derivative LP 3008. All mutations diminished swimming motility. When each mutant was co-inoculated with the parental strain on soybean plants cultivated in vermiculite either at field capacity or flooded, their competitiveness differed according to the flagellin altered. ΔfliCI-II mutants were more competitive, occupying 64-80% of the nodules, while ΔfliC1-4 mutants occupied 45-49% of the nodules. Occupation by the nonmotile double mutant decreased from 55% to 11% as the water content of the vermiculite increased from 85% to 95% field capacity to flooding. These results indicate that the influence of motility on competitiveness depended on the water status of the rooting substrate.     

Inactivation of Campylobacter jejuni flagellin genes by homologous recombination demonstrates that flaA but not flaB is required for invasion.

The role of the Campylobacter jejuni flagella in adhesion to, and penetration into, eukaryotic cells was investigated. We used homologous recombination to inactivate the two flagellin genes flaA and flaB of C. jejuni, respectively. Mutants in which flaB but not flaA is inactivated remain motile. In contrast a defective flaA gene leads to immotile bacteria. Invasion studies showed that mutants without motile flagella have lost their potential to adhere to, and penetrate into, human intestinal cells in vitro. Invasive properties could be partially restored by centrifugation of the mutants onto the tissue culture cells, indicating that motility is a major, but not the only, factor involved in invasion.     

Sporulation of Tricarboxylic Acid Cycle Mutants of Bacillus subtilis

A mutant of Bacillus subtilis 168 lacking aconitase (EC 4.2.1.3) was found to be blocked at stage 0 or I of sporulation. Although adenosine triphosphate levels, which normally decrease in tricarboxylic acid cycle mutants at the completion of exponential growth, could be maintained at higher levels by feeding metabolizable carbon sources, this did not permit the cells to progress further into the sporulation sequence. When post-exponential-phase cells of mutants blocked in the first half of the tricarboxylic acid cycle were resuspended with an energy source in culture fluid from post-exponential-phase wild-type B. subtilis or Escherichia coli, good sporulation occurred. The spores produced retained the mutant genotype and were heat stable but lost refractility and heat stability several hours after their production.     

The cucurbit pathogenic bacterium Acidovorax citrulli requires a polar flagellum for full virulence before and after host-tissue penetration

Acidovorax citrulli causes seedling blight and bacterial fruit blotch of cucurbits. Previous reports demonstrated the contribution of type IV pili (T4P) to A. citrulli virulence and to systemic infection of melon seedlings. Microfluidic flow-chamber assays demonstrated the involvement of T4P in surface adhesion and biofilm formation, whereas polar flagella did not appear to contribute to either of these features. On the other hand, a transposon mutant impaired in the biosynthesis of polar flagella was identified in screens for reduced virulence of an A. citrulli mutant library. Further characterization of polar flagellum mutants confirmed that A. citrulli requires a polar flagellum for full virulence on melon plants. Foliage and stem inoculation experiments revealed that polar flagella contribute to A. citrulli virulence and growth in planta at both pre- and post-host-tissue penetration. Interestingly, light microscope observations revealed that almost all A. citrulli wild-type cells extracted from the xylem sap of stem-inoculated melon seedlings remained motile, supporting the importance of this organelle in virulence and colonization of the host vascular system. We also report a negative effect of polar flagellum impairment on T4P-mediated twitching motility of A. citrulli and discuss a possible co-regulation of these two motility machineries in this bacterium.     

Properties of a Bacillus subtilis mutant able to sporulate continually during growth in synthetic medium

Several mutants of Bacillus subtilis were isolated which sporulate continually during exponential growth in glucose medium. The spdA1 mutation, responsible for the continual sporulation of one of the mutants, mapped near thr. When an exponentially growing culture of a strain containing spdA1 was maintained at essentially constant turbidity, 5% of the viable cells contained heat-resistant spores. The continual sporulation depended on the stringent response since it was absent in spdA relA double mutants. Genetic and biochemical analysis indicated that the continual sporulation of spdA1 strains was associated with a lower specific activity of pyruvate carboxylase, which limited the rate of oxaloacetate synthesis from glucose via pyruvate and thereby the supply of compounds depending on the citrate cycle, especially aspartate. Therefore, the mild stringent response caused by the spdA1 mutation seems to result from a partial deficiency of aspartyl-tRNA which may exert its sporulation-initiating effect during a limited time interval in each growth cycle. A mutant blocked in fumarase activity (citG) behaved similarly. It grew only slowly in glucose medium because much of the limiting oxaloacetate was wasted for the excretion of fumarate. The mutant produced little aspartate and sporulated at a high frequency in glucose medium, even in the presence of glutamate; the sporulation was again prevented by aspartate or malate or by introduction of the relA marker into the strain.     

Regulation of polar surface structures in Caulobacter crescentus: Pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors

Summary A large number of Caulobacter mutants resistant to DNA or RNA phages were isolated. These phage-resistant mutants exhibited phenotypic variations with respect to cell motility and sensitivity to other phages.The majority of the mutants was resistant to both DNA and RNA phages tested. In addition, these mutants were either motile or non-motile. The analysis of spontaneous revertants from these mutants indicated that a single mutation is involved in these phenotypic variations. Other mutants were resistant to RNA phages and only to a certain DNA phage tested, and were also motile or non-motile.Several temperature-sensitive phage-resistant mutants were also isolated. One of them, CB13 ple-801, exhibited the wild type phenotype when grown at 25°C. However, at a higher temperature (35°C), the mutant cells became non-motile and resistant to both DNA and RNA phages. These phenotypes seem to be attributed to the concommitant loss of flagella, pili and phage receptors. In other respects (cell growth and morphology, and asymmetric stalk formation), CB13 ple-801 was normal at 35°C. The spontaneous revertants from CB13 ple-801 simultaneously regained the wild type phenotypes in all respects.It is suggested that a single mutation pleiotropically affects the formation of flagella, pili and phage receptors.