The role of core and accessory type IV pilus genes in natural transformation and twitching motility in the bacterium Acinetobacter baylyi

ABSTRACT The ubiquitous species Pseudomonas stutzeri has type IV pili, and these are essential for the natural transformation of the cells. An absolute transformation-deficient mutant obtained after transposon mutagenesis had an insertion in a gene which was termedpilT. The deduced amino acid sequence has identity with PilT of Pseudomonas aeruginosa (94%), Neisseria gonorrhoeae (67%), and other gram-negative species and it contains a nucleotide-binding motif. The mutant was hyperpiliated but defective for further pilus-associated properties, such as twitching motility and plating of pilus-specific phage PO4. [3H]thymidine-labeled DNA was bound by the mutant but not taken up. Downstream of pilT a gene, termedpilU, coding for a putative protein with 88% amino acid identity with PilU of P. aeruginosa was identified. Insertional inactivation did not affect piliation, twitching motility, or PO4 infection but reduced transformation to about 10%. The defect was fully complemented by PilU of nontransformable P. aeruginosa. When thepilAI gene (coding for the type IV pilus prepilin) was manipulated to code for a protein in which the six C-terminal amino acids were replaced by six histidine residues and then expressed from a plasmid, it gave a nonpiliated and twitching motility-defective phenotype in pilAI::Gmr cells but allowed transformability. Moreover, the mutant allele suppressed the absolute transformation deficiency caused by the pilT mutation. Considering the hypothesized role of pilT + in pilus retraction and the presumed requirement of retraction for DNA uptake, it is proposed that the pilT-independent transformation is promoted by PilA mutant protein either as single molecules or as minimal pilin assembly structures in the periplasm which may resemble depolymerized pili and that these cause the outer membrane pores to open for DNA entry.

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Type IV Pilus Biogenesis, Twitching Motility, and DNA Uptake in Thermus thermophilus: Discrete Roles of Antagonistic ATPases PilF, PilT1, and PilT2

Applied and Environmental Microbiology ◽

10.1128/aem.03218-13 ◽

2013 ◽

Vol 80 (2) ◽

pp. 644-652 ◽

Cited By ~ 18

Author(s):

Ralf Salzer ◽

Friederike Joos ◽

Beate Averhoff

Keyword(s):

Natural Transformation ◽

Thermophilic Bacteria ◽

Bacterial Species ◽

Thermus Thermophilus ◽

Dna Uptake ◽

Twitching Motility ◽

Type Iv Pilus ◽

Content Type ◽

Ecological Diversification ◽

Type Iv

ABSTRACTNatural transformation has a large impact on lateral gene flow and has contributed significantly to the ecological diversification and adaptation of bacterial species.Thermus thermophilusHB27 has emerged as the leading model organism for studies of DNA transporters in thermophilic bacteria. Recently, we identified a zinc-binding polymerization nucleoside triphosphatase (NTPase), PilF, which is essential for the transport of DNA through the outer membrane. Here, we present genetic evidence that PilF is also essential for the biogenesis of pili. One of the most challenging questions was whetherT. thermophilushas any depolymerization NTPase acting as a counterplayer of PilF. We identified two depolymerization NTPases, PilT1 (TTC1621) and PilT2 (TTC1415), both of which are required for type IV pilus (T4P)-mediated twitching motility and adhesion but dispensable for natural transformation. This suggests that T4P dynamics are not required for natural transformation. The latter finding is consistent with our suggestion that inT. thermophilus, T4P and natural transformation are linked but distinct systems.

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Role of the Eikenella corrodens pilALocus in Pilus Function and Phase Variation

Journal of Bacteriology ◽

10.1128/jb.183.1.55-62.2001 ◽

2001 ◽

Vol 183 (1) ◽

pp. 55-62 ◽

Cited By ~ 20

Author(s):

Maria T. Villar ◽

Rona L. Hirschberg ◽

Michael R. Schaefer

Keyword(s):

Phase Variation ◽

S Phase ◽

Related Phenomenon ◽

Twitching Motility ◽

Type Iv Pilus ◽

Eikenella Corrodens ◽

Type Iv ◽

And Function ◽

Phase Variants

ABSTRACT The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolateE. corrodens VA1. Earlier work defined the pilAlocus which includes pilA1, pilA2,pilB, and hagA. Both pilA1 andpilA2 predict a type IV pilin, whereas pilBpredicts a putative pilus assembly protein. The role ofhagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilAmutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated forpilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation ofhagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.

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Mucin inhibitsPseudomonas aeruginosabiofilm formation by significantly enhancing twitching motility

Canadian Journal of Microbiology ◽

10.1139/cjm-2013-0570 ◽

2014 ◽

Vol 60 (3) ◽

pp. 155-166 ◽

Cited By ~ 10

Author(s):

Cecily L. Haley ◽

Cassandra Kruczek ◽

Uzma Qaisar ◽

Jane A. Colmer-Hamood ◽

Abdul N. Hamood

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iv Pili ◽

Dependent Manner ◽

Twitching Motility ◽

Strain Pao1 ◽

Concentration Dependent Manner ◽

Dependent Effect ◽

Type Iv ◽

Biofilm Development

In Pseudomonas aeruginosa, type IV pili (TFP)-dependent twitching motility is required for development of surface-attached biofilm (SABF), yet excessive twitching motility is detrimental once SABF is established. In this study, we show that mucin significantly enhanced twitching motility and decreased SABF formation in strain PAO1 and other P. aeruginosa strains in a concentration-dependent manner. Mucin also disrupted partially established SABF. Our analyses revealed that mucin increased the amount of surface pilin and enhanced transcription of the pilin structural gene pilA. Mucin failed to enhance twitching motility in P. aeruginosa mutants defective in genes within the pilin biogenesis operons pilGHI/pilJK-chpA-E. Furthermore, mucin did not enhance twitching motility nor reduce biofilm development by chelating iron. We also examined the role of the virulence factor regulator Vfr in the effect of mucin. In the presence or absence of mucin, PAOΔvfr produced a significantly reduced SABF. However, mucin partially complemented the twitching motility defect of PAOΔvfr. These results suggest that mucin interferes with SABF formation at specific concentrations by enhancing TFP synthesis and twitching motility, that this effect, which is iron-independent, requires functional Vfr, and only part of the Vfr-dependent effect of mucin on SABF development occurs through twitching motility.

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Two direct gene targets contribute to Clp-dependent regulation of type IV pilus-mediated twitching motility in Lysobacter enzymogenes OH11

Applied Microbiology and Biotechnology ◽

10.1007/s00253-018-9196-x ◽

2018 ◽

Vol 102 (17) ◽

pp. 7509-7519 ◽

Cited By ~ 9

Author(s):

Jiaojiao Chen ◽

Danyu Shen ◽

Benard Omondi Odhiambo ◽

Dan Xu ◽

Sen Han ◽

...

Keyword(s):

Twitching Motility ◽

Type Iv Pilus ◽

Lysobacter Enzymogenes ◽

Type Iv ◽

Direct Gene

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A Short Protocol for Gene Knockout and Complementation in Xylella fastidiosa Shows that One of the Type IV Pilin Paralogs (PD1926) Is Needed for Twitching while Another (PD1924) Affects Pilus Number and Location

Applied and Environmental Microbiology ◽

10.1128/aem.01167-18 ◽

2018 ◽

Vol 84 (18) ◽

Cited By ~ 7

Author(s):

Prem P. Kandel ◽

Hongyu Chen ◽

Leonardo De La Fuente

Keyword(s):

Natural Transformation ◽

Gene Knockout ◽

Xylella Fastidiosa ◽

Twitching Motility ◽

Wild Type ◽

Content Type ◽

Overlap Extension Pcr ◽

Type Iv ◽

Overlap Extension ◽

Pilin Subunit

ABSTRACT Twitching motility is one of the major virulence factors of the plant-pathogenic bacterium Xylella fastidiosa, and it is mediated by type IV pili (TFP) that are present at one of the cell poles. Genome analysis of X. fastidiosa showed the presence of at least four paralogs of the gene pilA, which encodes the TFP major pilin subunit. However, whether all of these paralogs have a functional role in TFP structure and function is unknown. Here, using a short and reliable protocol based on overlap extension PCR and natural transformation, deletion mutants of two pilA paralogs (pilA1 PD1924 and pilA2 PD1926) were generated in two X. fastidiosa subsp. fastidiosa strains, WM1-1 and TemeculaL, followed by assessment of twitching motility and biofilm formation. Deletion of pilA2 caused loss of twitching motility, whereas deletion of pilA1 did not influence twitching motility but caused hyperpiliation and extended distribution of TFP along the sides of the cell. Loss of twitching motility due to pilA2 deletion was restored when a wild-type copy of the pilA2 gene was added at a neutral site in the genome of mutants in both wild-type backgrounds. This study demonstrates that PCR templates generated by overlap extension PCR can be successfully used to rapidly generate gene knockouts and perform genetic complementation in X. fastidiosa, and that twitching motility in X. fastidiosa is controlled by regulating the transcription of the major pilin subunit, pilA2. IMPORTANCE The bacterial plant pathogen Xylella fastidiosa causes incurable diseases in multiple hosts, including grape, citrus, and blueberry. Historically restricted to the Americas, it was recently found to cause epidemics in olives in Italy and to infect other hosts in Europe and Asia. In this study, we report a short protocol to create deletion and complemented mutants using fusion PCR and natural transformation. We also determined the distinct function of two pilin paralogs, the main structural component of TFP involved in twitching motility, which allows this bacterium to move inside the xylem vessels against the flow. One of the paralogs is needed for twitching movement, whereas the other does not have an effect on motility but influences the number and position of TFP. Since twitching motility is fundamental for the virulence of this xylem-limited bacterium, this study contributes to the understanding of the regulation of virulence by this pathogen.

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Identification and Characterization of a Novel Competence Gene,comC, Required for DNA Binding and Uptake inAcinetobacter sp. Strain BD413

Journal of Bacteriology ◽

10.1128/jb.180.6.1592-1595.1998 ◽

1998 ◽

Vol 180 (6) ◽

pp. 1592-1595 ◽

Cited By ~ 22

Author(s):

Caroline Link ◽

Sandra Eickernjäger ◽

Dirk Porstendörfer ◽

Beate Averhoff

Keyword(s):

Electron Microscopy ◽

Dna Binding ◽

Natural Transformation ◽

Leader Sequence ◽

Wild Type ◽

Type Iv Pilus ◽

Competence Gene ◽

Type Iv ◽

Identification And Characterization

ABSTRACT A gene (comC) essential for natural transformation was identified in Acinetobacter sp. strain BD413. ComC has a typical leader sequence and is similar to different type IV pilus assembly factors. A comC mutant (T308) is not able to bind or take up DNA but exhibits a piliation phenotype indistinguishable from the transformation wild type as revealed by electron microscopy.

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Biological role of Actinobacillus pleuropneumoniae type IV pilus proteins encoded by the apf and pil operons

Veterinary Microbiology ◽

10.1016/j.vetmic.2018.08.006 ◽

2018 ◽

Vol 224 ◽

pp. 17-22 ◽

Cited By ~ 1

Author(s):

Feng Liu ◽

Wei Peng ◽

Ting Liu ◽

Haixu Zhao ◽

Kang Yan ◽

...

Keyword(s):

Actinobacillus Pleuropneumoniae ◽

Biological Role ◽

Type Iv Pilus ◽

Type Iv

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Type IV pilus gene homologs pilABCD are required for natural transformation in Actinobacillus actinomycetemcomitans

Gene ◽

10.1016/s0378-1119(03)00620-6 ◽

2003 ◽

Vol 312 ◽

pp. 249-255 ◽

Cited By ~ 25

Author(s):

Ying Wang ◽

Wenyuan Shi ◽

Weizhen Chen ◽

Casey Chen

Keyword(s):

Natural Transformation ◽

Actinobacillus Actinomycetemcomitans ◽

Type Iv Pilus ◽

Type Iv

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Pleiotropic Role of Quorum-Sensing Autoinducer 2 in Photorhabdus luminescens

Applied and Environmental Microbiology ◽

10.1128/aem.00398-06 ◽

2006 ◽

Vol 72 (10) ◽

pp. 6439-6451 ◽

Cited By ~ 40

Author(s):

Evelyne Krin ◽

Nesrine Chakroun ◽

Evelyne Turlin ◽

Alain Givaudan ◽

François Gaboriau ◽

...

Keyword(s):

Quorum Sensing ◽

Innate Immune ◽

Photorhabdus Luminescens ◽

Twitching Motility ◽

Autoinducer 2 ◽

Type Iv ◽

Integrative Process ◽

Dose Dependent

ABSTRACT Bacterial virulence is an integrative process that may involve quorum sensing. In this work, we compared by global expression profiling the wild-type entomopathogenic Photorhabdus luminescens subsp. laumondii TT01 to a luxS-deficient mutant unable to synthesize the type 2 quorum-sensing inducer AI-2. AI-2 was shown to regulate more than 300 targets involved in most compartments and metabolic pathways of the cell. AI-2 is located high in the hierarchy, as it controls the expression of several transcriptional regulators. The regulatory effect of AI-2 appeared to be dose dependent. The luxS-deficient strain exhibited decreased biofilm formation and increased type IV/V pilus-dependent twitching motility. AI-2 activated its own synthesis and transport. It also modulated bioluminescence by regulating the synthesis of spermidine. AI-2 was further shown to increase oxidative stress resistance, which is necessary to overcome part of the innate immune response of the host insect involving reactive oxygen species. Finally, we showed that the luxS-deficient strain had attenuated virulence against the lepidopteran Spodoptera littoralis. We concluded that AI-2 is involved mainly in early steps of insect invasion in P. luminescens.

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