UNIVERSIDADE ESTADUAL PAULISTA - UNESP
FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS
CÂMPUS DE JABOTICABAL
IDENTIFICATION AND CHARACTERIZATION OF ISXax2, A
TN3-LIKE TRANSPOSABLE ELEMENT POTENTIALLY
RELATED WITH THE VIRULENCE AND PATHOGENICITY OF
Xanthomonas citri subsp. citri
Rafael Marini Ferreira
Biólogo
2014
UNIVERSIDADE ESTADUAL PAULISTA - UNESP
FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS
CÂMPUS DE JABOTICABAL
IDENTIFICATION AND CHARACTERIZATION OF ISXax2, A
TN3-LIKE TRANSPOSABLE ELEMENT POTENTIALLY
RELATED WITH THE VIRULENCE AND PATHOGENICITY OF
Xanthomonas citri subsp. citri
Rafael Marini Ferreira
Orientador: Prof. Dr. Jesus Aparecido Ferro
Coorientador: Dr. Alessandro de Mello Varani
Tese apresentada à Faculdade de Ciências
Agrárias e Veterinárias – Unesp, Câmpus de
Jaboticabal, como parte das exigências para a
obtenção do título de Doutor em Agronomia
(Genética e Melhoramento de Plantas)
2014
Ferreira, Rafael Marini
F383i Identification and
characterization
of
ISXax2,
a
TN3-like
transposable element potentially related with the virulence and
pathogenicity of Xanthomonas citri subsp. citri. / Rafael Marini
Ferreira. – – Jaboticabal, 2014
x, 63 p. : il. ; 29 cm
Tese (doutorado) - Universidade Estadual Paulista, Faculdade de
Ciências Agrárias e Veterinárias, 2014
Orientador: Jesus Aparecido Ferro
Coorientador: Alessandro de Mello Varani
Banca examinadora: José Belasque Júnior, Marcos Tulio de
Oliveira, Maria Teresa Marques Novo Mansur, Maria Célia Bertolini
Bibliografia
1. Xanthomonas citri. 2. Transglicosilase Lítica de Mureína. 3.
SSTT 4. Transposon tipo TN3. 5. Efetores TAL. I. Título. II.
Jaboticabal-Faculdade de Ciências Agrárias e Veterinárias.
CDU 632.23:634.31
Ficha catalográfica elaborada pela Seção Técnica de Aquisição e Tratamento da Informação –
Serviço Técnico de Biblioteca e Documentação - UNESP, Câmpus de Jaboticabal.
DADOS CURRICULARES DO AUTOR
Rafael Marini Ferreira – nascido em Monte Alto, SP no dia 04 de maio de
1984, graduou-se em Ciências Biológicas (Modalidade Licenciatura e Bacharelado)
na Universidade Estadual Paulista “Júlio de Mesquita Filho” - Campus de Jaboticabal
em 2005. Trabalhou desde o primeiro semestre de 2002 até o presente momento no
Departamento de Tecnologia da Unesp de Jaboticabal, atuando nas áreas de
pesquisas proteômicas, clonagem, expressão heteróloga de proteínas, mutação sítio
dirigida e transposição genética em Xanthomonas citri subsp. citri. Desenvolveu
projeto nos anos de 2005 e 2006 intitulado “Correlação entre método de capturarecaptura e predação de ninhos artificiais para a estimativa populacional de
Didelphis albiventris” no Departamento de Zootecnia da Unesp de Jaboticabal.
Obteve título de Mestre em agronomia (Genética e Melhoramento de Plantas) pela
Universidade Estadual Paulista “Júlio de Mesquita Filho” - Campus de Jaboticabal no
ano de 2009, com projeto intitulado “Secretoma Da Bactéria Fitopatogênica
Xanthomonas citri subsp. citri”. Trabalhou por 6 meses no sequenciamento de
amostras de DNA no departamento de Tecnologia da FCAV-UNESP (atual CREBIO)
utilizando o sequenciador ABI Prism 3100. Foi bolsista de doutorado sanduíche
CAPES – PDEE (Processo BEX 2684/11-0), atuando na Universidade da California
Riverside (UCR) no departamento de Bioquímica sob supervisão do Prof. Dr. Li Fan,
na área de expressão, purificação e cristalização de proteínas de Xanthomonas citri
subsp. citri durante um ano (2011-2012). Obteve êxito na construção, indução e
purificação de quatro das referidas proteínas, porém as mesmas não cristalizaram.
Direcionou sua pesquisa para estudos em transposição genética nos anos de 2013 e
2014, tendo desenvolvido esta Tese de Doutorado no Departamento de Tecnologia
da FCAV-UNESP até o presente momento.
EPÍGRAFE
“Quem luta com monstros deve velar por que,
ao fazê-lo, não se transforme também em
monstro. E se tu olhares, durante muito tempo,
para um abismo, o abismo também olha para
dentro de ti.”
Friedrich Nietzsche
DEDICATÓRIA
Dedico esta tese aos meus pais, meu irmão,
meu amor e ao meu avô.
AGRADECIMENTOS
Ao Professor Doutor Jesus Aparecido Ferro pela orientação e ao Doutor
Alessandro de Mello Varani pela coorientação.
À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
pela bolsa de doutorado e doutorado sanduíche concedida ao autor.
Ao Professor Doutor Mick Chandler e ao Professor Doutor Leandro M. Moreira
pelas correções e sugestões dedicadas a este trabalho.
Aos Professores Doutores Manuel Victor Franco Lemos e Vitor Fernandes O.
De Miranda, à Professora Doutora Janete Apparecida Desidério e ao Doutor Tiago
Santana Balbuena pela composição da banca de qualificação.
Aos Professores Doutores José Belasque Júnior e Marcos Tulio de Oliveira, e
às Professoras Doutoras Maria Teresa Marques Novo Mansur e Maria Célia Bertolini
pela composição da banca e pelas correções e sugestões dedicadas a esta tese.
Ao Professor Doutor Jesus Aparecido Ferro e à Professora Doutora Maria
Inês Tiraboschi Ferro pela utilização do laboratório de bioquímica e biologia
molecular do departamento de Tecnologia da FCAV UNESP de Jaboticabal para a
realização dos experimentos e utilização de reagentes e equipamentos.
À aluna de iniciação científica Amanda C. P. de Oliveira pela parceria e ajuda
na realização dos experimentos.
À todas as pessoas maravilhosas que trabalham no LBM pela amizade e
atenção. São tantos amigos verdadeiros que fiz que nem caberiam todos os nomes
aqui. Acho que sei porquê é tão difícil deixar esse laboratório... ;-p.
A todos os meus amigos próximos, que tornaram o desenvolvimento dessa
tese algo muitas vezes divertido e leve, apesar dos percalços e dos medos
crescentes.
Ao Johnnie Walker Gold Label e a todos os amigos Macanudo, Gurkha,
Padilla e Cohiba, que tiveram que durar todos esses anos.
À minha família, que sempre esteve ao meu lado e ao amor da minha vida,
que é tão parte de mim que até deixei de lembrar que era tão necessário. Mas a
gente aprende.
Ao meu afilhadinho, que é a coisa mais linda do mundo!
8
SUMMARY
Page
RESUMO………………………………………………………………………….………….ix
ABSTRACT…………………………………………………………………………………...x
1 INTRODUCTION .................................................................................................... 11
2 LITERATURE REVIEW ............................................................................................. 13
3 MATERIALS AND METHODS ................................................................................17
3.1 Bioinformatics tools............................................................................................. 17
3.2 Overlap extension PCRs ..................................................................................17
3.3 Cloning and Xac Transformation ........................................................................ 18
3.4 In vivo pathogenicity test ..................................................................................19
3.5 ISXax2 Transposition.......................................................................................... 19
4 RESULTS .................................................................................................................. 21
4.1 Tn3-family elements in the pXAC64 plasmid and the distribution of their specific
TIRs among the Xanthomonads ............................................................................... 21
4.2 Chromosomal transposon derivatives ..............................................................26
4.3 Passenger Genes: Effector proteins, Mlt and MltB............................................. 27
4.4 MICs and TALEs................................................................................................. 29
4.5 Role of Mlt in pathogenicity as a hypersensitive response and pathogenicity
gene .......................................................................................................................... 36
4.6 Transposition of ISXax2 ...................................................................................38
5 DISCUSSION .........................................................................................................40
5.1 MICs and their relationship with emergence of Xanthomonas pathogenesis...40
5.2 Passenger Genes and their role for bacterial evolution .....................................40
5.3 Mlt role .............................................................................................................41
6 CONCLUSIONS ........................................................................................................ 43
7 REFERENCES .......................................................................................................44
8 SUPPLEMENTARY MATERIAL....................................................................................52
9
IDENTIFICATION AND CHARACTERIZATION OF ISXax2, a TN3-LIKE
TRANSPOSABLE ELEMENT POTENTIALLY RELATED WITH THE VIRULENCE
AND PATHOGENICITY OF Xanthomonas citri subsp. citri
RESUMO – O Cancro Cítrico é uma importante doença que afeta diversas espécies
de citros. Tal doença resulta da infecção pela bactéria Xanthomonas citri subsp. citri
(Xac), transmitida pela água, contato ou propagação de folhas infectadas. Estudos
comparativos têm mostrado que uma das características mais intrigantes do gênero
Xanthomonas é a extraordinária plasticidade do genoma e sua arquitetura altamente
mosaica. Isso se deve principalmente a rearranjos genômicos envolvendo diversas
ilhas genômicas (GI), sequências de Inserção (IS), e alguns eventos de inserção de
profagos. A anotação de IS de alta qualidade em conjunto com a genômica
comparativa revelaram um transposon completo da família TN3 no genoma de
Xanthomonas citri (ISXax2), contendo diversos genes passageiros efetores do SSTT,
incluindo a altamente conservada transglicosilase lítica de mureína (mlt), uma
proteína auxiliar do SSTT. Inserção aleatória de transposons foi utilizada para gerar
o mutante cromossomal mltB e mutação sítio-dirigida foi utilizada para gerar o
mutante plasmidial mlt. Usando ISXax2 de Xac como exemplo demonstramos
que a cópia plasmidial de mltB (mlt) era funcional e requerida para gerar sintomas in
vivo. Ensaios de transposição demonstraram taxas para ISXax2 com uma frequência
de cerca de 1.65 x 10-5. Neste estudo nós usamos uma mistura de abordagens
experimentais e de bioinformática para elucidar o papel e comportamento desse
potencial elemento transponível no plasmídeo de Xac. Demonstramos que a
estrutura completa foi capaz de sofrer transposição e que Cassetes de Inserção
Móveis (MICs) contendo efetores TAL também poderiam ser mobilizados. Estes
resultados mostram forte evidência que em Xac e espécies relacionadas, genes de
virulência e patogenicidade podem se espalhar através de um mecanismo de
transposição do tipo TN3, usando para tanto plasmídeos conjugativos como vetores
intercelulares. Propomos que este processo é um agente chave, modulando a
virulência bacteriana e a hospedeiro-especificidade neste grupo de fitopatógenos.
Keywords: Xanthomonas citri, Transglicosilase Lítica de Mureína, Transposon Tipo
TN3, Cassetes de Inserção Móveis, Efetores TAL
1
IDENTIFICATION AND CHARACTERIZATION OF ISXax2, a TN3-LIKE
TRANSPOSABLE ELEMENT POTENTIALLY RELATED WITH THE VIRULENCE
AND PATHOGENICITY OF Xanthomonas citri subsp. citri
ABSTRACT - Citrus canker (CC) is an important disease affecting many citrus
species. It can result from infection by Xanthomonas citri subsp. citri (Xac) and can
be transmitted by water, contact or propagation of infected leaves. Comparative
studies have shown that one of the most intriguing features within the Xanthomonas
genus is the extraordinary genome plasticity and highly mosaic architecture. This is
largely the consequence of genomic rearrangements involving several genomic
islands (GI), insertion sequences (IS), and few prophages insertion events. High
quality IS annotation coupled with comparative genomics revealed a complete
transposon of the Tn3 family in the Xanthomonas citri genome (ISXax2), with several
T3SEs passenger genes, including a highly conserved lytic murein transglycosylase
(mlt), a helper protein of the T3SS. Random transposon insertion was used to
generate XmltB and site directed mutagenesis was used to generate the mlt
mutant. Using ISXax2 from Xac as an example we demonstrated that the plasmid
copy of mltB (mlt) was indeed functional and required to generate symptoms in vivo.
Transposition assays showed transposition rates for ISXax2 with a frequency of
about 1.65 x 10-5. In this study we have used a mixture of bioinformatic and
experimental approaches to address the role and behavior of such potentially
transposable element. We showed that the entire structure was capable of
transposition and that MICs (Mobile Insertion Cassettes) containing TAL effectors
could also be mobilized. These results provide strong evidence that, in the case of
Xac and closely related species, pathogenicity and virulence genes can be spread by
a Tn3-like transposition mechanism, using conjugative plasmids as intercellular
vectors. We propose that this process is a key agent to modulate the bacterial
virulence and the host-specificity in this group of plant pathogens.
Keywords: Xanthomonas citri, Murein Lytic Transglycosylase, TN3-Like Transposon,
Mobile Insertion Cassettes, TAL Effectors
11
1 INTRODUCTION
Citrus canker (CC) is an important disease affecting many citrus species
(GOTTWALD, 2000). It results from infection by Xanthomonas citri subsp. citri and
can be easily transmitted by water, by contact or by propagation of infected leaves
(BRUNINGS; GABRIEL, 2003). The disease is endemic in many parts of the world
and has a severe economic impact on the citrus producing industry (SOARES et al.,
2010). CC involves a complex network of interactions implicating bacterial type II
(T2SS) and type III (T3SS) secretion systems and biofilm formation (RYAN et al.,
2011). T3SS efficiently transports effector proteins (T3SEs) from the bacterium into
the plant cell periplasm (SILVA et al., 2002). The effector proteins interact with
various host cell factors and interfere with cell growth and the defense response
causing hyperplasia and necrosis, which lead to cell lysis.
The genome sequence of Xanthomonas citri subsp. citri strain 306 (Xac)
pathotype A, a major causal agent of CC, was fully assembled over a decade ago
(SILVA et al., 2002). Moreover, other four different CC related species were partially
or completely sequenced recently; X. fuscans subsp. aurantifolii strains B and C,
(MOREIRA et al., 2010), X. citri subsp. citri strain A(w) 12879 (JALAN et al., 2013),
and X. axonopodis pv. citrumelo strain F1 (JALAN et al., 2011). Comparative studies
have shown that one of the most intriguing features within the Xanthomonas genus is
the extraordinary genome plasticity and highly mosaic architecture. Almost all of
these bacteria have a single circular chromosome that ranges in size from 4.8 Mb to
5.3 Mb, with a GC content of more than 60% and with a similar gene content in all
species, but whole-genome alignments have revealed a large number of inversions,
translocations and insertions or deletions. Further, at least one member of the genus,
X. albilineans, with a
genome of 3.7 Mb, seems to represent a lineage that is
undergoing genome reduction, which is thought to be related to its nearly exclusive
existence within the xylem of its host, sugarcane. Similarly, X. fastidiosa, with a
genome of ~2.7 Mb, is found only in association with host plant xylem or with the
insect vector that transmits it. Other vascular pathogens such as X. campestris pv.
campestris and X. oryzae pv. oryzae do not show similar genome reduction, and this
12
may reflect their lifestyle, which is not limited to growth in the xylem but can involve
the colonization of seed surfaces and growth on dead plant parts in the soil (RYAN et
al., 2011). This genome plasticity is largely the consequence of genomic
rearrangements involving several genomic islands (GI), insertion sequences (IS), and
few prophages insertion events (VARANI et al., 2013) (LIMA et al., 2008)
(MONTEIRO-VITORELLO et al., 2005). Interestingly, the closely related xylemlimited plant pathogen Xylella fastidiosa (Xf), which is transmitted by an insect vector,
in an opposite way of Xanthomonads, exhibits only relics of ISs but carries a
prophage load accounting for up to 10% of the genome size (VARANI et al., 2013).
Xac and Xf have a common ancestor (LIMA et al., 2005; VARANI et al., 2009) and
share a number of chromosome regions largely encoding housekeeping functions.
Since the virulence and pathogenicity factors carried by Xac and Xf are different,
these traits were probably acquired after divergence of the two bacterial lines
(SETUBAL et al., 2005). It is possible that many current differences between
Xanthomonas spp. and Xf resulted from acquisition by Xanthomonas of new genes
associated with IS elements and conjugative plasmids leading to an increase of up to
50% in its genome size compared to Xf (Xac is roughly 2,5 Mb greater than Xf) (LU et
al., 2008; VARANI et al., 2009). This difference may be related to the fact that
Xanthomonas exhibit a wider range of lifestyles, including colonization of leaf
surfaces and growth on dead plant material spread over the soil (RYAN et al., 2011).
This suggests that the gene loss is under negative selection, and the processes that
add genes are under positive selection, following the trend observed in the
prokaryotic world. An explanation might be that there is a constant pressure to lose
genes by gene deletion mutations, whereas the appearance of new genes only
occurs as an adaptation to a new lifestyle (SNEL et al., 2002). Indeed different
genomic analyses over the past years have revealed that several Xanthomonas spp.
genes related to pathogenesis and virulence, such as the T3SS and T3SE genes, are
located in plasmids and/or bordered by IS elements (NOEL et al., 2003; LAIA et al.,
2009; MOREIRA et al., 2010).
13
2 LITERATURE REVIEW
The T3SS proteins, as observed in other pathosystems, are an important part
of Xanthomonas pathogenesis (ROSSIER et al., 1999; BÜTTNER; HE, 2009). They
create a bridge between the bacterium and the plant host cell permitting injection of
bacterial T3SEs and other components (GALÁN; COLLMER, 1999; CORNELIS;
GIJSEGEM, VAN, 2000). It is interesting to note that many T3SEs in different
Xanthomonas species are often flanked by short (<100 bp) inverted repeat
sequences (IRs) (BOCH; BONAS, 2010). This type of genetic structure is typical of
Insertion Sequence-related Mobile Insertion Cassettes (MIC) (PALMENAER et al.,
2004; SIGUIER et al., 2012) and it has been suggested that these effector modules
may have been transmitted by lateral gene transfer (BOCH; BONAS, 2010). MICs do
not encode their own transposases, but can be activated by a cognate transposase
from an intact and related IS (VARANI et al., 2011; SIGUIER et al., 2014).
The T3SEs found in the MICs structures encodes to Transcription ActivatorLike Effectors (TALE). They are found in a number of Xanthomonads, representing a
class of DNA binding proteins, which modulate host gene expression (BOCH;
BONAS, 2010). TALEs are injected into eukaryotic host plant cells through the T3SS
and serve as transcription factors which act on host genes to the benefit of the
pathogen (GRAU et al., 2013). The host DNA-specificity is determined by conserved
tandem repeats, usually 34 amino acids long, including a Repeat-Variable Diresidue
(RVD) at position 12 and 13, each of which recognizes a given nucleotide (BOCH et
al., 2009; GRAU et al., 2013). TALEs are therefore potent pathogenicity proteins,
often targets for host-specific suppression of virulence (SHIOTANI et al., 2007; LIN et
al., 2011).
Previous studies performed by LAIA et al. (2009) using Random Transposon
Insertion Mutagenesis in Xac generated several thousand mutants with altered
virulence and pathogenicity when inoculated in planta. Among those genes,
XacmltB mutant showed decreased symptoms (water-soaking and hyperplasia) and
yet the pathogen still possessed another highly conserved copy of the same gene
(mlt) in the plasmid pXAC64. The role of mlt is not known and the fact that
14
bioinformatics analysis performed by us showed that this gene was contained in a
complete transposable structure led us to wonder about its role
in
Xac’s
pathogenesis and evolution.
Laia and co-authors previously suggested that mltB may be important for
T3SS apparatus formation and, consequently, for the Xac virulence (LAIA et al.,
2009).
The product of the conserved mltB gene is a lytic murein transglycosylase
unrelated to the T3SE proteins with orthologues in a number of bacteria including
Ralstonia and Pseudomonas. Interestingly, among the known X. oryzae species, the
mltB gene is only present in strain BLS256 (Table S3). In view of its probable
enzymatic function and the presence of a signal peptide it has been suggested that
MltB is secreted into the periplasm where it remodels the peptidoglycan layer
facilitating assembly of the T3SS (NOEL et al., 2003). MltB shares 63% sequence
identity to HopAJ1 from Pseudomonas syringae pv. tomato DC3000 (LAIA et al.,
2009; POTHIER et al., 2011). Although HopAJ1 is not a type III secretion system
substrate, it probably enables the type III secretion system to penetrate the
peptidoglycan layer in the bacterial periplasm and deliver virulence proteins into host
cells (OH et al., 2007). In Xac, it appears to be expressed specifically during infection
and its deletion reduces the severity of CC (LAIA et al., 2009). This conservation
suggests that an important selective pressure is operating to preserve the mltB/mlt
within these structures (Transposons), at least in Xanthomonads.
High quality IS annotation coupled with comparative genomics revealed a
complete transposon of the Tn3 family in the Xac genome. Tn3 family members are
very diverse in size and structure, ranging from 3,000 bp to more than 25,000 bp. All
include a transposase (Tnp) of the DDE motif class (OHTA et al., 2002) and a sitespecific recombinase system as well as passenger genes for functions such as
antibiotic and heavy metal resistance (e.g. mercury or copper - Tn5393) (CHIOU;
JONES, 1993) which have been sprayed for many years on vegetable and fruit crops
to limit the spread of plant pathogenic bacteria and fungi (VOLOUDAKIS et al., 2005).
Shorter derivatives have also been identified which encode only the
transposase and are devoid of the resolvase (e.g. ISVsa19, ISShfr9, ISBusp1,
IS1071D) (GIOIA, DI et al., 1998). These therefore resemble bacterial ISs (SIGUIER
15
et al., 2014). They transpose by replicative replicon fusion generating a cointegrate
with a copy of the transposon in direct orientation at each junction between the donor
and target replicons. Transposition terminates by recombination between the two
directly repeated Tn copies at a particular site, the res site, in a reaction catalysed by
a transposon-encoded site-specific recombinase (Figure 1). In many Tn3 family
members, this is a serine recombinase, the resolvase (TnpR). In others, it may be a
single tyrosine recombinase related to phage integrases (TnpI) or a pair of
recombinases (TnpT and TnpS).
Figure 1. Scheme depicting Tn3 family transposition mechanism by replicative
replicon fusion (GRIFFITHS et al., 1993).
In this study we used a mix of bioinformatics tools and experimental
procedures to investigate the role and behaviour of these Tn3 family transposable
elements in Xac. Murein Lytic Transglycosylase (mlt) is a passenger gene inside this
transposon and its role in Xac’s pathogenicity is unclear.
Our goal is to generate using site-directed mutagenesis Xacmlt, a truncated
plasmid mutation in Murein Lytic Transglycosylase, address its role in the virulence
and pathogenicity of Xac when inoculated in planta (Citrus sinensis) and analyse
16
whether virulence and pathogenicity genes like mlt and T3SE are spread by a Tn3Like transposition mechanism to other organisms.
Completion of those goals would lead to the assumption that this process is a
key agent in bacterial virulence modulation and host-specificity in this group of plantpathogens.
17
3 MATERIALS AND METHODS
3.1 Bioinformatics tools
Inverted Repeats (IRs) and Insertion Sequences (ISs) were searched using
ISfinder database (https://www-is.biotoul.fr). Gene sequences were aligned using the
algorithm Basic Local Alignment Search Tool nucleotide (Blastn) with standard
parameters, except for low complexity regions filter deactivated and word size 7
(ALTSCHUL et al., 1990).
The Molecular Phylogenetic analysis of the TALEs genes was performed using
sequences found in completely sequenced Xanthomonas genomes by the Maximum
Likelihood method. The alignment gaps between the different conserved tandem
repeats of each sequence were considered in the analysis and filled with Ns. The
evolutionary history was inferred by using the Maximum Likelihood method based on
the General Time Reversible model (KUMAR et al., 2004). The bootstrap consensus
tree
was
inferred
with
500
replicates
(FELSENSTEIN,
1997).
Branches
corresponding to partitions reproduced in less than 50% bootstrap replicates were
collapsed. Initial trees for the heuristic search were obtained automatically by
applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances
estimated using the Maximum Composite Likelihood (MCL) approach, and then
selecting the topology with superior log likelihood value. A discrete Gamma
distribution was used to model evolutionary rate differences among sites (3
categories (+G, parameter = 1.0586)). The analysis involved 122 TALEs sequences,
including a total of 8174 positions in the final dataset. Evolutionary analyses were
conducted in MEGA6 (TAMURA et al., 2013).
3.2 Overlap extension PCRs
mltB mutation was carried out according to Laia (LAIA et al., 2009). Sitedirected mutagenesis through overlap extension PCR was used to generate
truncated mlt copies according to Lee (2010). The following primers were designed: A
(5’-GTGTGGATCCGGGCATTCTGACCGCCAT-3)’,
GCTCGGTGATTTTCCGTCTGCAGGATCT-5),
B
C
(3’(5’-
18
GACGGAAAATCACCGAGCTGCAGCAGAG-3)’
and
D
(3’-
AAGGTGAGGGATATCCCCGATCAGGGGGCCCGC-5’). The template DNA used for
PCR was XAC’s pXAC64 plasmid DNA cloned in a Lawrist cosmid in E. coli DH10B.
Primer A was designed with a restriction site for BamHI and D with ApaI.
Primers B and C were designed in a way that they are complementary to each other,
so after a first round of PCR, the template used for the second PCR is no longer the
cosmid DNA, but the product generated by the first PCR. This way a big portion of
the gene is deleted.
PCR reactions were performed as follows: 100 ng DNA, 200 μM dNTPs, 0.5
μM Forward primer, 0.5 μM Reverse primer, 1X Phusion GC buffer, 3% DMSO, 1.0
unit/50 μl PCR of Phusion DNA polimerase (New England BioLabs inc ®).
Thermocycling conditions were used according to manufacturer and primers
annealing temperatures. After reaction, PCR products were loaded in 0,8% agarose
gels.
PCR products were purified using Wizard SV Gel and PCR Clean-up System
Kit (Promega ®).
3.3 Cloning and Xac Transformation
The 362 bp amplification product was digested with ApaI and BamHI (New
England BioLabs inc ®) according to manufacturer specifications. Suicide vector
derived from pKNG101, pOKI was used to delete the wildtype mlt gene from Xac
(KANIGA et al., 1991) and was also digested under the same conditions. Both
digestion products were ligated by T4 DNA ligase (New England BioLabs inc ®) and
electroporated in Xac (AMARAL et al., 2005).
Xac transformed cells were spread in Nutrient Agar plates (0.5% Peptone,
0.3% beef extract, 1.5% agar, 0.5% NaCl, pH adjusted to 6.8) containing 50 g/mL
Spectinomycin. SpecR colonies were selected and spread in Nutrient Agar plates
containing 5% sucrose and 50 g/mL Spectinomycin. The colonies sensitive to
sucrose and Spectinomycin were selected as potential mutants, since pOK1 vector
confers resistance to Spectinomycin and sensitivity to sucrose at first, which is then
lost considering its suicide nature (KANIGA et al., 1991).
Candidate mutant Xac colonies plasmid DNA was extracted and sequenced to
19
confirm mlt gene mutation.
3.4 In vivo pathogenicity test
After confirmation, mltB and mlt mutants were cultivated in Nutrient
Agar plates without antibiotics for 72 hours. Xac mutant cells were harvested, its
concentration measured in autoclaved bi-distilled water with a spectrophotometer at
600 nm until O.D. 0.3 (108 CFU/mL – colony forming units per mL).
Bacterial suspensions were inoculated on the under side of sweet orange
leaves (Citrus sinensis) using a hypodermic syringe without the needle.
Inoculated plants were kept in quarantine green house under the same
temperature, humidity and light cycle. Symptoms were observed and photographed
at high resolution starting in day 3 up to day 21 after inoculation (LAIA et al., 2009).
3.5 ISXax2 Transposition
Transposition experiments were performed according to Aubert et al (2006)
and Johnson (1984). LB broth (1% tryptone, 0.5% yeast extract, 1% NaCl) was used
for all matings together with the following antibiotics according to the selection
needed: 20 g/mL Nalidixic acid, 50 g/mL Rifamycin, 20 g/mL Kanamycin, 10
g/mL Gentamycin and 200 g/mL Streptomycin.
The recipient strain used (Female) was E. coli XA103 (NalR RifR) and the male
was E. coli DH10B containing Xac plasmid construction in Lawrist cosmid together
with pOX38Gm (StreptR KmR GmR).
LB plates with respective antibiotics were cultivated for both male and female
cells at 37 °C overnight. Four separate male colonies were used to start 4 precultures
in 10 mL LB liquid broth with antibiotics and one female culture in the same
conditions. The cells were cultivated overnight at 37 °C at 180 rpm. O.D (600 nm)
was measured and adjusted to 0.05 to start new cultures in 50 mL LB broth (without
antibiotics) flasks for both male and female. O.D. was tracked until the cultures
reached 0.5. The male flasks were then kept in the same temperature but without
shaking for 1 hour. The female flask was rediluted to O.D. = 0.2 and kept under
vigorous shaking.
15 mL of each male culture and 7 mL of female culture were carefully mixed
20
and kept at 37 °C for 1 hour and 15 minutes without shaking for the conjugation
experiment. The flasks were then placed on ice and vigorously vortexed for 1 minute,
in order to stop the mating.
The mating mixture was then centrifuged at 4,000 rpm for 10 minutes. Cells
were ressuspended in 400 L of LB. 10X Serial dilutions were made in 500 L
eppendorf® tubes until 10-7 and 100 L of each dilution was plated in LB solid broth
containing the following antibiotics:
Ex-conjugants: Nal20, Rif50, Gm10; Co-integrates: Nal20, Rif50, Gm10, Km20; negative
control: Nal20, Rif50, Gm10 (E. coli DH10B containing only Xac plasmid).
Transposition rate was measured according to the formula: co-integrates/exconjugants.
All experiments were made in 5 triplicates, standard deviation and average
were calculated.
The mating out experiment is summarized in figure S2.
In order to check whether transposition was actually occurring, several primers
were designed for the following passenger genes: mlt - (5’-GTGTGGATCCGGGCAT
TCTGACCGCCAT-3)’, (3’-AAGGTGAGGGATATCCCCGATCAGGGGGCCCGC-5’);
MIC (XACb0015) Position 14.450 – 15.082 – (5’-GAGGGTTGCCAGGATCACCC-3’),
(3’-TGTCGAACGAACCTTCGGTT-5’); MIC (XACb0015) Position 17.233 – 17.833 –
(5’- TTTCCAGTGCCACTCCCACC-3’), (3’- CTCCATCAACCATGCGAGCT-5’) and
RES site Position 9509 – 9735 – (5’-GGTGGCGGCTTCCAGGTATT-3’), (3’- CCGT
CACTCCACGTATTTCG-5’).
PCR reactions were performed as described above. Primers annealing
temperature were adjusted using a gradient thermocycler.
PCR products for the genes mlt, 5’ end of pthA3, 3’end of pthA3 (MICs) and
Res site using cointegrates extracted DNA as template were loaded in 0,8% Agarose
gels and gel electrophoresis were conducted at 80 V during 1 hour. 5 L of PCR
products were loaded in gel. PCR using Pre-transposition pOX38Gm template DNA
was also performed as negative control.
21
4 RESULTS
4.1 Tn3-family elements in the pXAC64 plasmid and the distribution of their
specific TIRs among the Xanthomonads
Plasmid pXAC64 and closely related plasmids are widely distributed in
Xanthomonads (MOREIRA et al., 2010; POTHIER et al., 2011). This has presumably
occurred by conjugal transfer between these bacteria since they encode a transfer
operon (a type IV secretion system; T4SS) with a relaxase gene related to trwC of
plasmid R388 and traI of the classical conjugative F plasmid. Noël et al. (2003)
identified a region in the sequence of pXAC64 (SILVA et al., 2002) from X. citri in
which the xopE2 (avrXacE3) effector gene and a lytic murein transglycosylase gene
(mlt) were associated with three genes related to transposable elements (TE). This
structure is flanked by inverted repeat sequences.
The nature of the TE related genes in this case was not addressed by the
authors. We have revisited this question in the course of our ongoing studies of Xac
pathogenesis. We find not only that the flanking inverted repeat sequences
correspond to the highly conserved terminal inverted repeat (TIR) sequences of
members of the large Tn3 family of transposons, but that the TE-like associated
genes are also typical of Tn3 family members. We have provisionally called this
potentially transposable structure, ISXax2 (Figure 2 A).
The ISXax2 TIR sequences are shown in Figure 2 B. They are 92 bp long with
72 bp identity. Although this is rather long for Tn3 TIRs, several other members of the
family are also known to carry TIRs of this length. Tn3 TIRs generally exhibit blocks
of A residues as does the left ISXax2 TIR (Figure 2 B). The tips of the TIR begin with
the sequence GAGGG instead of GGGGG, which is more common for this family.
Noteworthy that the ISXax2 full length TIR of 93 bp are also found flanking a
probable peptidase gene (see below) in a unique sequenced beta proteobacteria
genome, the cucurbit pathogen Acidovorax avenae subsp. citrulli strain AAC00-1,
accounting eight different occurrences (Table S1). However, except for this example,
full length TIRs associated with the Xac Tn3 transposon are restricted to the gammaproteobacteria, mainly in Xanthomonas species. There are at least 264 different
22
occurrences in Xanthomonadales (Table S1). Moreover these TIRs are generally part
of ISXax2 relatives, putative MIC elements as well as several examples of solo TIRs.
Figure 2. (A) Structural organization of ISXax2 located in X. axonopodis pv. citri str.
306 plasmids pXAC64. The resolution site is located between the tnpS and
tnpT genes. The terminal inverted repeats (TIRs) are represented as black
triangles. (B) Comparison of TIR sequences from ISXax2 and other Tn3-like
transposable elements bearing the GAGGG tips, showing shared sequence
similarities. Source: ISPa43 from Pseudomonas aeruginosa, ISStma18 from
Stenotrophomonas maltophilia strain D457, ISTin1 from Thiomonas
intermedia strain K12, and ISXca3 from X. campestris pv. vesicatoria
plasmid pXCV183.
23
Interestingly, a 20 kb potential Tn3 family transposon, ISPa43 (ISfinder), from
Pseudomonas aeruginosa also carries GAGGG at its tips and shows significant
sequence identities with its Xanthomonas ISXax2 TIRs (Figure 2 B), although the
eighteen passenger genes (mostly involved in phosphonate and phosphinate
metabolism, Table S2) are completely unrelated to those of ISXax2. In addition, TIRs,
including the GAGGG tips, are also found in three different Tn3 family transposons
which are also unrelated to ISXax2: ISStma18 (ISfinder), from Stenotrophomonas
maltophilia strain D457, ISTin1 (ISfinder), from Thiomonas intermedia strain K12 and
ISXca3 (ISfinder) from X. campestris pv. vesicatoria plasmid pXCV183 (Figure 2 B).
The ISStma18 TIRs are 35 bp long and 34 bp identical, flanking a 28 kb element,
which carries eighteen passenger genes (mostly related to the heavy metal
metabolism) (Table S2). The ISTin1 TIR are 32bp long and 32 bp identical, but the
element is 6,122 bp long and encodes only the transposition genes (TnpA, TnpS and
TnpT). The ISXca3 TIR are the shortest ones with 28 bp long and 23 bp identical.
Moreover ISXca3 is 11,523 bp long, encoding seven passenger genes, including a
potential UDP-N-acetylglucosamine kinase, tRNA(fMet)-specific endonuclease, and a
putative virulence associated protein (Table S2).
ISXax2 carries three transposition-related ORFs. XACb0008 (Figure 2 A)
resembles a Tn3 transposase gene, whose closest relative in the ISfinder database
is the transposase of ISPa43 from P. aeruginosa (62% identity and 78% similarity at
the protein level). It is worthy mentioning that ISPa43 is also the element which
carries the most similar TIR to those of ISXax2. Significant similarities over the entire
length of the protein are also observed with transposases of elements from
Shewanella (ISSod9), Acinetobacter (ISAcsp1), Arthrobacter (ISArsp6), Azospirillum
(ISAzs17), Salmonella (ISSwi1), Nostoc (ISNpu13) and the mercury resistance
transposon Tn5044 from X. campestris.
The second orf, XACb0009, (Figure 2 A), also shows high similarity with an
equivalent gene in ISPa43 (49% identity and 65% similarity). This is a tyrosine
recombinase with similarities to phage integrases and the XerCD proteins. This gene
appears frequently in the Xanthomonadales and is often referred to as “cointegrate
resolution protein S” (TnpS) (YANO et al., 2013). ISXax2 XACb0009 is related to
TnpS of the P. putida mt2 plasmid pWW0 transposon Tn4651 (54% identity and 69%
24
similarity) but not to the tyrosine recombinases, TnpI, found in elements such as
Tn4430 from Bacillus (NICOLAS et al., 2010).
The third orf, XACb0010 (Figure 2 A), is related to the cointegrate resolution
protein T (TnpT) of the toluene catabolic transposon, Tn4651 (YANO et al., 2013)
(43% identity and 61% similarity). Note that TnpS and TnpT are oriented divergently
in both Tn4651 and ISXax2. For Tn4651, the intergenic region between the divergent
tnpS and tnpT genes is 188 bp and includes two short palindromes, IRL/IRR and
IR1/IR2, within a 136 bp functional resolution recombination site (YANO et al., 2013)
(the Tn3 family res site). IRL and IRR are 13bp long and separated by a 7 bp region,
the core site, at which recombination occurs (YANO et al., 2013). The tnpS-tnpI
intergenic region in ISXax2 is 193 bp and also includes two palindromic sequences of
14bp with one mismatch and a potential core of 5bp (Figure 2 A) suggesting that this
may represent the resolution or res site. ISXax2 therefore exhibits all the
characteristics of a functional Tn3 family transposon.
A structure with a similar organization of TnpA, TnpS, TnpI and res site can be
found in the related plasmid, pXap41, from X. arboricola pv. pruni isolated from a
variety of sources (Figure 3 A and B) (POTHIER et al., 2011). This pXap41 element
includes approximately 14 kb of additional DNA at its right end (Figure 2 B), which
contains a second TIR in the orientation of the left TIR followed by a series of
insertions of passenger genes. It also includes a different Tn3 family transposon
inserted close to the right TIR. The transposase gene of this Tn3 family derivative
has 84% identity and 90% similarity to the ISPsy30 transposase with two TIRs
flanking the transposase and a presumed resolvase gene. The entire structure is
flanked by 5 bp direct target repeats as expected for Tn3 family members. A partial
transposase of ISXac3 is also present on the right end next to the TIRR (Figure 3 B).
25
Figure 3. Structural organization of the Left (A) and Right (B) Ends of the ISXax2
relatives structures found in other Xanthomonas related species.
26
4.2 Chromosomal transposon derivatives
In addition to the pXAC64 copy, ISXax2 relatives are found in the chromosome
of at least ten different Xanthomonas spp. strains. Most of these are citrus-related
pathogens. They carry the characteristic tnpA, tnpS and tnpT genes and the res site.
However, the right-end is highly variable (Figure 3 B). Interestingly, most of these
variations result from insertion of other ISs and acquisition of other passengers
genes. For instance the XAC chromosome (Xac 306 chr) carries three identical
transposition-related genes, but this is probably inactive in transposition because it
lacks the left TIR (Figure 3 A). Interestingly, the extreme left region of 475bp including
the left TIR is located 335 kb away in another region of the chromosome and in an
inverted
orientation,
flanking
an
IS
from
IS3
sub-group
IS407
and
rhamnogalacturonan hydrolase B gene (rhgB) (Figure S1). The tip of the right TIR
has the sequence GAAGG, but the displaced left TIR has the characteristic GAGGG
tip.
X. citrumelo F1 (Figure 3 B), is a citrus pathogen causing bacterial
spot
disease and, at present, restricted to Florida/USA (JALAN et al., 2011). This strain
carries a copy of ISXax2 with flanking TIRs together with a second partial copy in an
inverted orientation located some 100kb away. This duplication might have occurred
by replicative transposition from the original location to a second location in the same
chromosome. Moreover the right end of Xac F1 chr A is longer, while the Xac F1 chr
B copy lacks the right TIR (Figure 3 B). The Xac F1 chr A carries a copy of IS404
(previously described in Burkholderia cepacia 249, where its presence increases
beta-lactamase gene expression (SCORDILIS et al., 1987)). Xac F1 chr A also
carries a disrupted copy of IS1389 (originally described in X. campestris pv.
amaranthicola and X. oryzae pv. oryzae). Interestingly these insertions are located
bordering a disrupted copy of the xopC effector (Figure 3 B). In addition, there is an
insertion of a full-length copy of IS1595 (previously described in X. campestris pv.
mangiferae indicae) to the right of tnpT and
a partial copy of ISXac5 originally
described in X. campestris pv. vesicatoria 85-10 close to the right TIR (Figure 3 B).
Two relatives of ISXax2 are found in X. campestris pv. vesicatoria 85-10
(Figure 3), a causal agent of pepper and tomato leaf spot. The first gene cluster (Xcv
85-10 chr A) is flanked by the conserved TIRs and contains the IS1595 copy inserted
27
to the right of tnpT and a partial copy of a second IS, ISXca5, is located next to the
right TIR. Interestingly the Xcv 85-10 first cluster structure carry a complete XopC
copy, which is disrupted on Xac F1 (Figure 3 B) whereas the second cluster (Xcv 8510 chr B) is significantly different. Its left end lacks the TIR whereas the right end
carries two additional passenger genes next to the xopE2 effector. In addition to the
absence of the left TIR, the tnpT has an in frame stop codon suggesting that the
entire structure is no longer transpositionally active (Figure 3 B).
The Xanthomonas citri subsp. citri strain Aw12879 (Xcc Aw12879) carries a
related ISXax2 copy resembling a chimera of the Xac 306 chr left end and ISXax2
right end structures (Figure 3 A and B) suggesting that a recombination event was
responsible for the origin of the Xcc Aw12879 copy. However, as observed for Xcv
85-10 second cluster, the Xcc Aw12879 tnpT has an in frame stop codon, thus
indicating that the element may be not functional (Figure 3 B).
The Xanthomonas axonopodis strain 29-1 carries two ISXax2 related copies
(Xac29-1 chr and Xac29-1 pXAC64). The Xac29-1 pXAC64 left end is identical to
that of ISXax2. However the Xac29-1 chr has in its right end the XopE2 and XopAI
effectors and two TIRs. This structure resembles a chimerical element derived from
those ISXax2 and Xac 306 chr, respectively (Figure 3 B), and again suggesting a
recombination event has occurred.
The draft sequences of two X. fuscans subsp. aurantifolii strains (XauB and
XauC), causal agents of type B and C cankers respectively, contain related structures
(Figure 3 A and B) but we are unable to determine whether these are intact (neither
sequence contains both flanking TIRs) or are located on the chromosome or on a
plasmid since the genome sequence of both strains are not yet complete.
4.3 Passenger Genes: Effector proteins, Mlt and MltB
The transposon-like structures described above were initially identified in
regions containing a number of T3SE genes involved in pathogenicity. The annotated
regions in Figure 3 A and B show that these occur as passenger genes within many
of the structures. One gene, mlt, encoding a lytic murein transglycosylase, initially not
considered to belong to the T3SE protein family repertoire (POTHIER et al., 2011), is
consistently found in these structures except for a single case from X. campestris pv.
28
vesicatoria 85-10 (second cluster) (Figure 3 A). Laia and co-authors (2009)
previously suggested that mltB may be important for T3SS apparatus formation and,
consequently, for the Xac virulence. This conservation suggests that an important
selective pressure is operating to preserve the mlt within these structures, at least in
Xanthomonads. It is noteworthy that the mlt homologues are also found outside the
ISXax2 structure in other genera such as of Pseudomonas and Ralstonia
(see
below).
Other genes known for their pathogenic role are the effector genes xopE2,
xopAI and xopC which have received much attention over the past few years, and
are considered central for virulence and pathogenicity (NOEL et al., 2003; THIEME et
al., 2007; XIE et al., 2011; JALAN et al., 2013).
The structures in Figure 3 A can be divided into two groups: an ISXax2 group
with a 429bp linker between the end of mlt and the beginning of the left TIR and a
second group including the xopE3 (avrXacE2) gene represented by the pXap41
structure. In these, xopE3 occurs 85 bp from the 3' end of mtl, and is followed by a
785bp linker. Members of this group retain the left TIR. The chromosomal derivative
of ISXax2 is particular in that it includes the 785bp and 85 bp linkers and xopE3, but
does not retain the TIR. Therefore these ISXax2 chromosomal copies are most likely
incapable of transmitting their xopE3 and mlt genes by transposition.
The major difference between the pXAC64 and Xac 306 chr ISXax2 copies is
that they carry two different T3SE genes. ISXax2 from pXAC64 includes xopE2, a
putative transglutaminase which acts on the plant cell plasma membrane, suggesting
a role in the virulence and in the suppression of the hypersensitive response of the
host (THIEME et al., 2007; LIN et al., 2011). The Xac 306 chr copy includes xopAI,
homologous to the virulence factors HopU1, HopO1-1 and HopO1-2 from
Pseudomonas syringae pv. Tomato, probably having a post-translational modification
function (NICAISE et al., 2013) (Figure 3 A and B). Interestingly both effectors share
about 200 bp at their 5' ends. The C terminal end of XopAI shows some similarity to
the arginine ADP-ribosyltransferase family (PF01129). The chromosomal ISXax2
copy also includes an additional 274 bp of non-coding DNA between XopAI and the
right TIR, whereas ISXax2 has 204 bp of non-coding DNA between XopE2 and the
29
right TIR. Other differences between the ISXax2 structures are much more complex
indicating a high level of recombination involved in their formation (Figure 3 B).
Both ISXax2 relatives from X. citrumelo F1 carry a disrupted copy of the XopC
effector (Figure 3 B). Previous experimental studies have indicated that the XopC
effectors are secreted and translocated into the plant cell (NOEL et al., 2003). We
suggest that the disrupted XopC copies found in the ISXax2 relatives from X.
citrumelo F1 result from recombination potentially mediated by a neighboring IS404
element (Figure 3 B).
Many of the other passenger genes carried by these structures are
also
related to virulence. For instance, the Xcv 85-10 second cluster carries a putative
secreted protein, presumably involved in virulence, and a pectin lyase gene
potentially capable of degrading the plant cell wall (IPR011050) (MAYANS et al.,
1997). Interestingly the XauC ISXax2 relative carries four genes probably involved in
cell metabolism (Figure 3 B). The first has a TauD/TfdA (IPR003819) domain, related
to taurine catabolism of dioxygenases, which in E. coli is used as a sulphur source
and expressed only under conditions of sulphate starvation (EICHHORN, 1997). The
second includes a nucleoside triphosphate hydrolase domain (IPR027417),
potentially capable of hydrolysis of the beta-gamma phosphate bond of a bound
nucleoside triphosphate (LEIPE et al., 2004). The third has an aminotransferase
class-III domain (IPR005814), and the forth has the oxoglutarate/iron-dependent
dioxygenase domain (IPR005123). Moreover the pXap41 has seven hypothetical
genes and some plasmid related genes such as parA, parC and repA as passenger
genes.
4.4 MICs and TALEs
One of the present areas of great interest stemming from the Xanthomonads
are the TALE genes. These have attracted attention by their simple and predictable
specific DNA sequence recognition readout which uses a number of repeating
peptide blocks some of which include a dipeptide with specific recognition properties
for a given base (BOCH et al., 2009; GRAU et al., 2013). This has led to their fusion
to other proteins to design and target the chimeric protein to specific DNA sequences
(in both prokaryotes and eukaryotes (BOCH et al., 2009)). In their original
30
Xanthomonad hosts, TALE proteins are transmitted to the host plant cell nucleus
where they activate or inactivate specific genes, thus contributing directly to virulence
and pathogenicity. Indeed a single amino acid substitution in the TALE sequence can
affect canker formation (SHIOTANI et al., 2007; LIN et al., 2011). However, the
mechanism of transcriptional activation by TAL effectors is still not fully understood
(GRAU et al., 2013).
Previous studies had revealed that members of the TAL gene family in
different Xanthomonas strains are flanked by 62 bp TIR and it was suggested that
these are transmitted as mobile cassettes. Two of these were identified on each of
the two Xac plasmids pXac33 (Ptha1 and Ptha2) and pXac64 (Ptha3 and Ptha4)
(BOCH; BONAS, 2010). All four genes are slightly different, and the main differences
are located on the repeating peptide blocks, but each is flanked by a pair of TIR with
the same sequence (92 bp long with 72 bp identity) as those of the Xanthomonasspecific TIR carried by ISXax2 (Figure 4). This suggests that the Tn3-like element
may provide transposition functions for these MIC elements.
Figure 4. Mobile Insertion Cassettes (MICs) associated to T3SE (TAL effectors) on
both Xac plasmids (pXAC33 and pXAC64). Red arrows indicate TAL
effectors. Black triangles indicate terminal inverted repeats (TIR) similar to
ISXax2.
31
Boch and Bonas (2010) previously described 113 known TALEs distributed
among different Xanthomonas species. In this study we analyzed these genes
together with newly sequenced TALE genes deposited in the public databases. In this
analysis we focused in TALEs distributed mainly in complete and fully sequenced
genomes, to verify their association with ISXax2 TIRs. A maximum likelihood tree
constructed using the full-length sequences of the 122 different TALEs considered in
this study are shown in Figure 5.
The tree topology shows two major groups: an ancient group composed of the
X. oryzae species (in light orange), and a group composed of the X.
axonopodis/campestris/citri species (in green) (Figure 5).
32
Figure 5. Molecular Phylogenetic analysis by Maximum Likelihood method of the
TALEs genes found in completely sequenced Xanthomonas genomes.
Each TALE gene are shown with their respective genomic coordinates (for
TALEs extracted from complete genomes) or Genbank Accession Number
(for TALEs not from complete genomes) inside the brackets. The red
diamonds represent the TALEs within MIC structures, the yellow triangles
represent the TALEs which have a SoloTIRs flanking one of their
extremities, and the blue circles represent the genomes which carry the
ISXax2 or relatives structures. The black arrows indicates where the Xac
33
TALEs are located on the tree, and the blue arrow indicate the TALE from X.
fuscans subsp. fuscan str. 4834-R plasmid pla, located over the oryzae
group. The analysis involved 122 TALEs sequences, including a total of
8174 positions in the final dataset.
Interestingly, the TALE located on the X. fuscans subsp. fuscans str. 4834-R
plasmid plc (Xff-plc) is grouped with axonopodis/campestris/citri species, whereas the
TALE located in the X. fuscans subsp. fuscans str. 4834-R plasmid pla (Xff-pla)
groups with the oryzae species. This suggests that the TALEs present in Xff-plc and
Xff-pla, which are a causal agents of common bacterial blight of bean (DARRASSE et
al., 2013), may have evolved independently from an X.axonopodis/campestris/citri
lineage for Xff-plc, and an X.oryzae lineage for Xff-pla.
The X. axonopodis/campestris/citri group is mostly formed by TALEs located in
plasmids. This group also carries all the ISXax2-related copies. The X. oryzae group
(represented principally by strains MAFF 311018, PXO99A, KACC 103331 and
BLS256) do not carry plasmids, and indeed their TALEs are exclusively located in the
chromosome. Moreover the X. oryzae group does not carry ISXax2 related
structures. In summary, the X. oryzae group TALEs represent the majority (87 or
72%) compared to those of the X. axonopodis/campestris/citri group (35 or 28%) in
the ratio of 19 to 2 (oryzae to axonopodis/campestris/citri, respectively) TALEs per
genome.
A considerable number of TALEs forming potential MICs structures were also
identified. A total
of
52
(42%)
potential
MICs,
26
for
each
group
of
axonopodis/campestris/citri and oryzae. Moreover 22 SoloTIRs were identified
flanking TALEs from oryzae group, and only 1 SoloTIRs was identified in
axonopodis/campestris/citri group.
In spite of the absence of ISXax2 relatives in the X. oryzae group, the PXO99A
strains carry partial structure similar to ISXca3 which share part of the same TIRs of
ISXax2. The ISXca3 from PXO99A has a TnpA and TnpS with 59% and 80% identity,
respectively to those in ISXax2 (Table S3). Interestingly, TnpA, TnpS and TnpT
forming the complete structure of ISXca3 is originally found in Xcc str. Aw12879
plasmid pXacw58. An additional and partial copy without TIRs is also found in Xcc str.
B100 (Table S3). Since the ISXca3 TIRs and transposition related genes shares a
considerable similarity to those on ISXax2, ISXca3 may be functional representative
34
of the MICs found in PXO99A strain. It is noteworthy that a partial ISXca3 relative
structure is also found in the genome of Pseudoxanthomonas spadix str. BD-a59
(Table S3).
The ISXax2 relative structures forming a potential Tn3 transposon are mostly
found in the chromosome and plasmids of Xanthomonas arboricola, axonopodis,
campestris and vesicatoria, whereas the ISXax2 TIRs from oryzae and oryzicola are
exclusively found in chromosomes forming potential MICs and SoloTIR (Figure 6).
Moreover, the X. oryzae and X. oryzicola genomes do not carry the ISXax2 element,
and not even isolated copies of the TnpA, TnpS, TnpT genes and res site in their
genomes.
In addition to those MICs associates with TALEs, we also identified MIC
structures associated with other presumed effector proteins in X. fuscans subsp.
fuscans strain 4834-R chromosome, and Acidovorax avenae subsp. citrulli strain
AAC00-1 (Table S2). These MICs carry genes encoding a YopJ (serine/threonine
acetyltransferase) domain gene (IPR005083; and GenBank Accessions: ABM33278
ABM33505, CDF61199 and CDF61902). This domain is involved in the signaling
pathways by blocking phosphorylation in eukaryotic cells and inhibits innate immune
signaling (MUKHERJEE et al., 2006; PAQUETTE et al., 2012). Xanthomonas
campestris pv. vesicatoria strains are known to possess four YopJ-like proteins,
AvrXv4, AvrBsT, AvrRxv, and XopJ, all effectors secreted by T3SS (RODEN et al.,
2004).
35
Xac29-1
Xac F1
Xcv 85-10
A. citrulli
Xcc
ATCC33913
Xcc B100
Xcc 8004
Xcc Aw12879
Xcc 306
Xoo POX99A
Xoo
MAFF311018
Xoo KACC
10331
Xoo BLS 256
Xa GPE PC73
Legend
Xcv pXCV183
IRL
IRR
IRL (incomplete)
IRR (incomplete)
ISXax2 relatives structures
TAL effector
T3SEs
mlt
Putative secreted protein
Xac pXAC64
Xac29-1 pXAC64
Xcc Aw12879
pXcaw58
Xff 4834-R pla
Xff 4834-R plc
Xap CFBP 5530
plasmid pXap41
Xc pXcB
Xac pXAC33
Legend
IRL
Xac29-1 pXAC33
IRR
IRL (incomplete)
IRR (incomplete)
Xag 8ra pXAG81
ISXax2 relatives structures
TAL effector
T3SEs
mlt
Tn3 related genes
Xa GPE PC73
Xag AG1 pAG1
Secreted protein
36
Figure 6. Structural representation of ISXax2 relatives and TAL effectors most
present on chromosomes on X. oryzae and oryzicola species (Top) and the
same structures located mostly on plasmids in X. arboricola, axonopodis,
campestris and vesicatoria.
4.5 Role of Mlt in pathogenicity as a hypersensitive response and pathogenicity
gene
To determine whether mlt is indeed part of the transposon-based arsenal of
pathogenicity genes and indeed a hypersensitive response and pathogenicity gene
(hrp), we generated mutants and tested their effects on the Citrus sinensis host.
Xac carries two mlt genes (XACb0007 and XAC3225): one within ISXax2 on
pXAC64 and a second, mltB associated with the chromosome ISXax2 relative
(Figure 3 A). The 425 amino acid MltB differs from Mlt by four silent mutations and a
single conservative amino change (Ser to Ala).
The plasmid mutant was generated by PCR site-directed mutagenesis to
generate an internal out of frame deletion between codon 193 and codon 369,3
resulting a truncated protein of 248 amino acids, but with a premature stop-codon
(Figure 7). The MltB mutant obtained by transposon mutagenesis has been
described (LAIA et al., 2009). In this mutant, a mini-Tn5 insertion is located at codon
39, resulting in a short N-terminal peptide (Figure 7). Although it proved relatively
easy to obtain each of the single mutants we were consistently unable to construct
the double mutant, suggesting that Mlt may be essential in Xac.
37
Figure 7. and mutants and their respective effects when
inoculated in Citrus sinensis at 108 CFU/mL. Wt stands for Wild type Xac,
mltB::Tn5 for Xac MltB chromosomal mutant and mlt for Xac plasmid
mutant.
38
The phenotypes of the individual mutants on Citrus sinensis leaves are shown
in Figure 7. Clearly, the plasmid mlt mutation () eliminates all symptoms of
infection even though the strain carries a wildtype copy of mltB, while the mltB mutant
() shows only a reduced level of symptoms. These results clearly show that
mlt plays a central role in infection of Citrus sinensis by Xac. Both mlt and mltB genes
contribute to pathogenesis as shown in the left side of each of the four panels but Mlt
single amino acid substitution (Alanine to Serine) greatly increases it’s catalytic
activity, thus explaining the complete lack of symptoms when is inoculated in
planta as opposed to symptoms. Thus, the plasmid mlt copy is more
important to Xac’s virulence and pathogenicity than mltB (chromosomal copy), but as
observed in Pseudomonas syringae pv. tomato DC3000, both genes might still have
a potential overlapping role in contributing to T3SS functions during infection.
4.6 Transposition of ISXax2
Little is known about the transposition events and behavior in Xac genomes.
Therefore in view of their potential role in the spread of CC pathogenicity
determinants, we examined the capacity of ISXax2 to undergo transposition using a
mating out assay in an E. coli host. The conjugative plasmid pOX38Gm (MAKRIS et
al., 1988) was introduced into E. coli DH10B carrying the Lawrist cosmid 1AD06. This
cosmid includes ~44 kb of pXAC64 spanning both ISXax2 and the MICs pthA3
(XACb0015, coordinates: 14,558 to 17,848). This strain was then used as a donor in
matings with E. coli XA103. Since ISXax2 does not itself carry a selectable marker
but should generate cointegrates between the transposon donor plasmid and the
conjugative pOX38Gm target plasmid, we selected for pOX38Gm-mediated transfer
of the ISXax2 present in the Lawrist cosmid (KmR). Selection was for Km (for the
cosmid), Gm (for pOX38Gm), Nal and Rif for the recipient strain. KmRGmRE.coli
XA103 (potential cointegrates) arose with a frequency of about 1.65 x 10-5 compared
to GmR colonies (ex-conjugants having received pOX38Gm alone).
PCR products for the genes mlt, 5’ end of pthA3, 3’end of pthA3 (MICs) and
Res site using cointegrates extracted DNA as template were loaded in Agarose gels
and showed that indeed Xac ISXax2 transposed to pOX38Gm plasmid, as did the
MIC and Res sites (Figure 8).
39
Figure 8. 0.8% Agarose gel electrophoresis conducted at 80 V during 1 hour. 5 L
PCR products of the following genes were loaded in gel: 1) Mlt; 2) 5’ end of
pthA3 (MIC); 3) 3’ end of pthA3 (MIC) and 4) Res site. Cointegrates
extracted plasmid DNA used as template for PCR. PCR using Pretransposition pOX38Gm template DNA showed no signs of amplification.
40
5 DISCUSSION
In this work we identified a transposable element, ISXax2 of the large Tn3
family from Xanthomonas citri, which plays an important role in virulence and
pathogenicity. We have also identified a number of related structures with variable
genetic organization in a variety of Xanthomonads. Although the left ends of these
structures fall into two relatively homogenous groups with two principal organizations,
the right ends are highly variable, showing evidence of recombination and
duplication.
5.1 MICs and their relationship with emergence of Xanthomonas pathogenesis
One of the most intriguing features of the bacterial genomes are the mobile
insertion cassette elements (MICs). These elements are present in a number of
bacterial genomes. For instance, previous studies based on Bacillus cereus, showed
that their MICs can be mobilized in trans by a cognate transposase (CHEN et al.,
1999), and moreover indicated that these MICs could have a number of roles such as
endopeptidase, antibiotic resistances or regulatory factors, thus contributing to
genome evolution and plasticity (PALMENAER et al., 2004). On Xac genome four
MICs related to the ISXax2 were identified. Interestingly, ISXax2 associated T3SEs
and TALEs genes represent at least 33% of all Xac T3SEs arsenal.
We also suggest that these MICs can be mobilized in trans by the ISXax2
transposase, corroborating the Boch and Bonas previous suggestion (2010), and
thus indicating that emergence of important pathogenicity traits on Xac are directly
related to ISXax2. Indeed this MICs structures are often spread among others
Xanthomonas related species, and their roles in the emergence of pathogenicity
should be considered.
5.2 Passenger Genes and their role for bacterial evolution
It was recently proposed that some Xanthomonadale T3SEs genes have been
subjected to a shuffling process, called “terminal reassortment”, in which the 5' end
has undergone “random” genetic fusions resulting in new chimeric T3SEs genes with
41
a variety of 3' ends (STAVRINIDES et al., 2006). The role of this T3SE reassortment
is presumably to generate a wide spectrum of novel chimeric proteins, which could
affect both bacterial virulence and host specificity. It is believed that the Lateral Gene
Transfer mediated by Mobile Genetic Elements such as ISs can act as a motor for
this process (STAVRINIDES et al., 2006). In this work we have shown evidence that
the transposition mediated by a Tn3-like element may be a key agent in the process
of spread and transmission of T3SEs, and may have a potential role as an agent of
“terminal reassortment”.
The other identified Tn3 family transposons which shares the ISXax2 TIRs
(GAGGG tips) are indeed related to the bacterial lifestyle, reflecting directly the
pathogenicity and virulence in gamma-proteobacteria. For instance, the phosphonate
and phosphinate genes found in ISPa43 from P. aeruginosa may have a central role
in pathogenicity. With abundance of key nutrients such as phosphate, P. aeruginosa
is capable to express a virulent and lethal phenotype, including swarming motility and
cytotoxicity (BAINS et al., 2012; ZABORIN et al., 2012). Other elements such as
ISStma18 from Stenotrophomonas maltophilia shows significant importance for
bacteria lifestyle. The Stenotrophomonas genus are multidrug resistant, and the
passenger genes from ISStma18 may also be related to the tolerance for high levels
of various toxic metals, such as Cd, Pb, Co, Zn, Hg, Ag, selenite, tellurite and uranyl
(PAGES et al., 2008; RYAN et al., 2009).
Because the genera Xanthomonas, Pseudomonas, and Stenotrophomonas
are from the gamma-proteobacteria group and share a close common ancestor,
lateral gene transfer mediated by these Tn3-like transposable elements probably
played a central role in their differentiation, shaping their life styles and hostspecificity.
5.3 Mlt role
We demonstrated that the mlt gene is clearly involved with the outcome of CC
symptoms. Interestingly, transcriptome and proteome experiments have shown that
both mlt and mltB genes are up-regulated when Xac is inoculated in different
susceptible hosts (Figure 9) (MURATA, 2013).
42
Figure 9. mlt, mltB and T3SE genes regulation in RNAseq and proteomics
experiments when Xac was inoculated in several resistant and susceptible
hosts in different days after inoculation (24, 48 and 72 hours), compared to
rich media (Nutrient Agar) as standard condition. Yellow triangles show fold
change > 2.
In summary the transposition by Tn3-like elements may be crucial during the
course of the Xanthomonads evolution, and our results will certainly lead to novel
insights related to the citrus canker
traits and their relationships with
Xanthomonas genome architecture and evolution.
the
43
6 CONCLUSIONS
In this study we have used a mixture of bioinformatic and experimental
approaches to address the role and behavior of a potential transposable element.
The Xac Tn3-like transposons also include T3SEs passenger genes together with a
highly conserved lytic murein transglycosylase gene (mltB). Using one of these,
ISXax2 from Xac, we demonstrated by mutation that the plasmid copy of mltB gene is
indeed functional and required to generate symptoms in the plant, as previously
suggested (LAIA et al., 2009). We also showed that the entire structure was capable
of transposition.
The analysis also revealed that the four Xac MICs, distributed between the
pXAC33 and pXAC64 plasmids carry TALEs passenger genes. These MICs are
flanked by the same Tn3-like repeats as the ISXax2 transposon. In several citrus and
related species, the TALEs in MIC structures are often found in plasmids, whereas in
the X. oryzae species these occur in the chromosome. Moreover we also
demonstrate that the Xac MICs could be mobilized by transposition functions
supplied in trans by the complete ISXax2 transposon. Since most X. oryzae species
members do not carry plasmids, this observation suggests that a plasmid borne
ISXax2 relative and TALEs MICs may have transiently exchanged genetic material
between X. citri/axonopodis/campestris and X. oryzae, and many of their TALEs may
have spread by transposition.
These results therefore provide strong evidence that at least in the case of
Xac and other closely related species, pathogenicity and virulence genes may have
been spread by a Tn3-like transposition mechanism and use conjugative plasmids as
intercellular vectors. We propose that this process is a key agent to modulate the
bacterial virulence and the host-specify in this group of plant pathogens.
44
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52
8 SUPPLEMENTARY MATERIAL
Table S1. List of occurrence of the ISXax2 full-length TIRs in Xanthomonadales
(gamma-proteobacteria) sequences deposited in GenBank. The genome
coordinates represent the orientation of each TIR. For identity values were
considered the IRR of ISXax2 as reference. These ISXax2 TIRs are also
found forming a potential MIC element in a unique beta-proteobacteria
genome, Acidovorax citrulli strain AAC00-1 a watermelon pathogen.
Genome or Plasmid identified
Xanthomonas axonopodis Xac29-1 pXAC64 (64,118 bp)
IR start
3989
12932
13749
17700
25197
52639
53945
58406
IR stop
4080
12842
13839
17609
25108
52729
54035
58315
%id
92/92
74/92
78/91
75/94
80/90
74/92
78/91
75/94
Xanthomonas axonopodis Xac29-1 (5,153,455 bp)
3802552
3811473
3814001
4151804
3802643
3811382
3813911
4151713
92/92
72/94
70/91
76/92
Xanthomonas axonopodis pv. citrumelo F1 (4,967,469 bp)
2452240
2558748
2438697
2452149
2558839
2438771
92/92
92/92
66/76
Xanthomonas campestris pv. vesicatoria 85-10 (5,178,466 bp)
662686
2480425
2603770
2610728
2614800
2764198
2776534
4369321
662630
2480345
2603802
2610693
2614710
2764272
2776443
4369401
51/58
66/85
30/33
32/36
75/92
66/76
92/92
67/83
3350
12294
14311
17959
25458
53668
54974
58823
3441
12204
14401
17868
25369
53758
55064
58732
92/92
74/92
78/91
75/94
80/90
74/92
78/91
75/94
2975223
2978700
2978784
2978963
3224781
3225044
3224960
3228521
2975303
2978785
2978756
2978872
3224872
3224959
3224988
3228441
66/85
69/86
26/29
90/92
90/92
69/86
26/29
66/85
93
8706
8796
14156
14246
17444
17354
40015
1
8797
8771
14247
14221
17353
17379
40105
75/94
77/92
24/26
76/92
24/26
76/92
24/26
83/91
455
545
82/91
Xanthomonas axonopodis pv. citri str. 306 pXAC64 (64,920 bp)
Acidovorax citrulli AAC00-1 (5,352,772 bp)
Xanthomonas fuscans subsp. fuscans str. 4834-R, pla (45,224 bp)
Xanthomonas citri pv. aurantifolii strain C340 PthC (4,876bp)
53
4325
4519
4278
4453
44/49
58/68
Xanthomonas citri plasmid pXcB (37,106 bp)
31463
35333
35527
31553
35286
35461
82/91
44/49
58/68
Xanthomonas fuscans subsp. fuscans str. 4834-R, plc (41,950 bp)
19199
21844
22338
26372
30746
31908
38186
41950
19108
21934
22247
26462
30837
31820
38276
41904
75/92
75/91
73/92
75/91
73/92
76/90
81/91
42/47
3263
3353
81/91
Xanthomonas axonopodis Xac29-1 pXAC33 (31,801 bp)
9094
17429
24927
28584
9004
17518
25018
28494
78/91
80/90
75/94
78/91
Xanthomonas axonopodis pv. citri str. 306 pXAC33 (33,700 bp)
5469
12968
16706
30836
5558
13059
16616
30746
80/90
75/94
78/91
78/91
Xanthomonas citri subsp. citri Aw12879 pXcaw58 (58,317 bp)
12492
22394
26247
27553
12582
22485
26156
27463
78/91
74/94
78/92
74/92
Xanthomonas citri pv. citri strain X0053 PthAW (4,559 bp)
460
4312
550
4221
78/91
74/94
Xanthomonas smithii subsp. citri pB3.7 (4,500 bp)
133
4378
223
4287
78/91
75/94
Xanthomonas smithii subsp. citri pthA-KC21 (4,070 bp)
109
3959
199
3867
78/91
75/95
Xanthomonas smithii subsp. citri pB3.1 (3,985 bp)
119
3767
209
3676
78/91
75/94
101
191
78/91
3912
4663
14711
14897
3821
4573
14802
14987
75/94
74/92
78/92
78/91
349
4198
439
4107
78/91
75/94
1
91
78/91
Xanthomonas campestris pv. armoraciae Hax3 (3,218 bp)
1
3218
91
3150
78/91
56/71
Xanthomonas citri apl3 (4,639 bp)
101
4562
191
4471
78/91
75/94
Xanthomonas citri apl2 (3,823 bp)
101
191
78/91
Xanthomonas citri apl1 (4,027 bp)
101
3950
191
3859
78/91
75/94
Xanthomonas vesicatoria avrBs4 (4,000 bp)
72
3912
162
3821
78/91
75/94
Xanthomonas vesicatoria plasmid pXV11 avrBs3 (4,366 bp)
374
4226
464
4135
78/91
75/94
Xanthomonas axonopodis pv. manihotis strain CFBP1851 pthBXam (10,736 bp)
Xanthomonas citri hssB3.0 gene for avirulence protein (3,718 bp)
Xanthomonas axonopodis pv. glycines strain AG1 plasmid pAG1 (15,143 bp)
Xanthomonas citri strain 3213 PthA (4,275 bp)
Xanthomonas campestris pv. armoraciae Hax (3,507 bp)
54
Xanthomonas campestris pv. campestris str. ATCC 33913 (5,076,188 bp)
1902124
2466511
2482712
2482622
2491914
2492195
3434576
4438478
1902190
2466591
2482622
2482650
2492005
2492272
3434666
4438566
57/68
67/82
72/91
26/29
76/92
60/84
74/91
75/91
Xanthomonas campestris pv. campestris str. B100 (5,079,002 bp)
1453050
2409940
2410221
2791168
2791078
1452960
2409863
2410130
2791078
2791106
74/91
60/84
76/92
73/91
26/29
Xanthomonas campestris pv. campestris str. 8004 (5,148,708 bp)
1476237
2419112
2419393
2496533
2496623
2512734
3137704
4496061
1476147
2419035
2419302
2496623
2496595
2512654
3137638
4496149
74/91
60/84
76/92
72/91
26/29
67/82
57/68
75/91
Xanthomonas fuscans subsp. fuscans str. 4834-R (4,981,995 bp)
1884500
1886283
2750592
2751922
4939801
4946417
4946455
4953056
1884596
1886240
2750676
2751831
4939891
4946327
4946423
4953122
74/97
40/45
69/85
74/92
76/92
75/91
31/35
59/68
Xanthomonas citri subsp. citri Aw12879 (5,321,499 bp)
1262821
3952635
4729175
1262911
3952544
4729084
71/91
72/94
76/92
Xanthomonas axonopodis pv. citri str. 306 (5,175,554 bp)
3807325
4143510
3807235
4143419
70/91
76/92
Xanthomonas campestris PthN (3,994 bp)
38
135
3579
3489
128
225
3488
3517
76/92
76/92
75/92
26/29
Xanthomonas oryzae pv. oryzae strain PXO99 type III effector AvrXa23 (6,452 bp)
100
754
5541
6195
9
844
5450
6285
72/92
72/91
73/92
74/91
321285
324403
559020
842065
1625939
1633509
1645151
1649608
1650262
1781506
1838885
2359265
2359919
2363552
2387282
2387936
2392130
2527364
2681275
2682578
2686432
2687086
2739451
321375
324314
559111
842003
1625971
1633420
1645242
1649518
1650353
1781425
1838816
2359175
2360010
2363461
2387191
2388026
2392039
2527426
2681206
2682668
2686341
2687176
2739513
73/92
72/91
73/92
51/63
31/33
65/91
72/92
73/91
72/92
65/82
57/71
73/91
73/92
73/92
73/92
72/91
72/92
51/63
58/72
74/91
72/92
71/91
51/63
Xanthomonas oryzae pv. oryzae PXO99 (5,240,075 bp)
55
2893362
2894665
2898519
2899173
4104892
4105546
4110333
4110987
4114733
4115387
4118731
4119385
2893293
2894755
2898428
2899263
4104801
4105636
4110242
4111077
4114642
4115477
4118640
4119475
58/72
74/91
72/92
71/91
72/92
72/91
73/92
74/91
72/92
72/91
73/92
72/91
Xanthomonas oryzae pv. oryzae strain C8 Avr/pthC8b (4,525 bp)
3617
4271
3526
4361
72/92
74/91
Xanthomonas campestris pv. malvacearum Avrb6 (3,856 bp)
314
3763
404
3672
76/92
75/94
1232038
1232692
1236438
1237092
1241876
1242530
2204911
2205565
2209623
2210920
2218495
2352097
2356288
2356941
2360576
2386917
2387571
3028589
3085100
3218732
3219386
3223441
3224095
3228882
3229536
3234212
3244797
3252366
4300513
4529444
4763458
4765690
1231948
1232783
1236348
1237183
1241786
1242621
2204821
2205656
2209533
2210989
2218433
2352188
2356197
2357032
2360486
2386826
2387661
3028658
3085181
3218641
3219476
3223350
3224185
3228791
3229626
3234121
3244886
3252334
4300575
4529353
4763547
4765600
74/91
73/92
74/91
72/92
72/91
72/92
74/91
73/92
74/91
59/72
51/63
72/92
73/92
73/92
73/91
73/92
72/91
57/71
65/82
73/92
74/91
73/92
74/91
73/92
73/91
71/92
65/91
31/33
51/63
73/92
72/91
72/92
3347
4001
8788
9442
13188
13842
17186
17840
3256
4091
8697
9532
13097
13932
17095
17930
72/92
72/91
73/92
74/91
72/92
72/91
73/92
72/91
1265943
1266597
2225929
2226583
2230641
2231938
2239521
2377246
2381013
2401087
2401741
2406648
1265853
1266688
2225839
2226674
2230551
2232007
2239482
2377337
2380923
2400996
2401831
2406604
72/91
73/92
74/91
71/92
74/91
59/72
36/40
72/92
73/91
73/92
72/91
40/45
Xanthomonas oryzae pv. oryzae MAFF 311018 (4,940,217 bp)
Xanthomonas oryzae pv. oryzae AvrXa27 (22,214 bp)
Xanthomonas oryzae pv. oryzae KACC 10331 (4,941,439 bp)
56
2407301
3035786
3092324
3223775
3224429
3229927
3230581
3234546
3252722
4307290
4534573
4767482
4770598
2407392
3035855
3092405
3223684
3224519
3229836
3230671
3234455
3252690
4307352
4534482
4767569
4770508
73/92
57/71
65/82
72/92
72/91
72/92
73/91
71/92
31/33
51/63
73/92
72/91
72/92
101536
112968
113058
101571
113058
113030
31/36
73/91
26/29
Xanthomonas arboricola pv. pruni str. CFBP 5530 plasmid pXap41 (41,102 bp)
1514
11847
15966
25065
1598
11812
16031
24975
70/86
31/36
57/69
74/92
Xanthomonas axonopodis pv. glycines strain 8ra plasmid pXAG81 (26,721 bp)
3912
4663
22797
3821
4573
22887
75/94
74/92
74/92
Xanthomonas campestris pv. campestris beta-galactosidase Gal35I (3,423 bp)
2801
2892
74/92
653841
1558706
1686668
1697100
2548617
2556024
653779
1558796
1686616
1697009
2548706
2555991
53/63
73/92
47/53
74/93
66/91
31/34
Xanthomonas oryzae pv. oryzicola Avr/Pth13 (3,170 bp)
2852
2761
74/93
Xanthomonas campestris pv. vesicatoria outer protein B (23,471 bp)
8848
8904
51/58
Xanthomonas campestris pv. vesicatoria avirulence protein AvrBsT (1,931 bp)
1395
1484
71/92
4
92
70/91
2246665
1840960
2246755
1840907
68/91
42/54
61
141
66/85
7897
7932
33/36
Xanthomonas campestris pv. vesicatoria plasmid pXCV183 (182,572 bp)
Xanthomonas oryzae pv. oryzicola BLS256 (4,831,739 bp)
Xanthomonas oryzae avirulence protein AvrXa10 (3,720 bp)
Xanthomonas albilineans GPE PC73 (3,768,695 bp)
Xanthomonas campestris pv. vesicatoria outer protein J (1,441 bp)
Xanthomonas albilineans str. GPE PC73, plasmid (24,837 bp)
Table S2. Tn3-like transposable elements with similar TIRs (GAGGG tips) to ISXax2.
List of the transposition and passenger genes identified in ISPa43 from
57
Pseudomonas aeruginosa (A), ISStma18 from Stenotrophomonas
maltophilia strain D457 (B), and ISXca3 from X. campestris pv. vesicatoria
plasmid pXCV183 (C). The grey rows indicate the transposition related
genes. The coordinates represent the position of each open reading frame
in the transposon itself.
A) ISPa43 :19,903 bp – Source: ISfinder
Locus Tag
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
Element ID
ISPa43_00001
ISPa43_00002
ISPa43_00003
ISPa43_00004
ISPa43_00005
ISPa43_00006
ISPa43_00007
ISPa43_00008
ISPa43_00009
ISPa43_00010
Coordinates
43..1,824
2,224..2,451
2,769..3,629
3,654..4,550
4,674..5,522
5,556..6,368
6,380..6,925
7,011..7,412
7,439..7,918
7,911..8,495
Length
1,782
228
861
897
849
813
546
402
480
585
Orientation
+
+
+
+
+
+
+
+
n/d
ISPa43_00011
8,495..9,616
1,122
+
n/d
n/d
n/d
ISPa43_00012
ISPa43_00013
ISPa43_00014
9,613..10,518
10,744..11,316
11,330..12,055
906
573
726
+
+
+
n/d
n/d
n/d
n/d
n/d
n/d
ISPa43_00015
ISPa43_00016
ISPa43_00017
ISPa43_00018
ISPa43_00019
ISPa43_00020
12,052..13,206
13,220..13,789
13,780..14,547
14,544..15,353
15,471..16,805
16,862..19,849
1,155
570
768
810
1,335
2,988
+
+
+
+
+
+
Predicted Product
Methyltransferase domain protein
hypothetical protein
ectoine hydroxylase
Phosphate-import protein PhnD precursor
Phosphate-import permease protein PhnE
Phosphate-import ATP-binding protein PhnC
phosphonate ABC transporter, permease protein PhnE
Phosphate-import permease protein PhnE
phosphonate C-P lyase system protein PhnG
Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase
subunit PhnH
Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase
subunit PhnI
Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P lyase
Putative phosphonates utilization ATP-binding protein PhnK
Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase
subunit PhnL
Alpha-D-ribose 1-methylphosphonate 5-triphosphate diphosphatase
Ribose 1,5-bisphosphate phosphokinase PhnN
Phosphoribosyl 1,2-cyclic phosphodiesterase
phosphonate utilization associated putative membrane protein
site-specific tyrosine recombinase XerD (TnpI)
Transposase, TnpA family (DDE domain)
B) ISStma18: 28,714 bp from Stenotrophomonas maltophilia D457 - NC_017671
Locus Tag
SMD_2129
SMD_2130
SMD_2131
Element ID
ISStma18_00001
ISStma18_00002
ISStma18_00003
Coordinates
84..1,142
1,465..1,857
1,929..3,233
Length
1,059
393
1,305
SMD_2132
SMD_2133
SMD_2134
SMD_2135
SMD_2136
SMD_2137
SMD_2138
SMD_2139
SMD_2140
SMD_2141
n/d
SMD_2142
SMD_2143
SMD_2144
SMD_2145
SMD_2146
SMD_2147
ISStma18_00004
ISStma18_00005
ISStma18_00006
ISStma18_00007
ISStma18_00008
ISStma18_00009
ISStma18_00010
ISStma18_00011
ISStma18_00012
ISStma18_00013
ISStma18_00014
ISStma18_00015
ISStma18_00016
ISStma18_00017
ISStma18_00018
ISStma18_00019
ISStma18_00020
3,230..4,726
4,723..7,884
7,930..8,286
8,379..8,780
9,152..9,661
9,654..10,901
10,924..11,244
11,244..14,369
14,407..15,597
15,594..16,871
17,170..17,337
17,575..19,971
19,968..21,938
22,127..22,567
22,605..24,251
24,226..25,527
25,566..28,550
1,497
3,162
357
402
510
1,248
321
3,126
1,191
1,278
168
2,397
1,971
441
1,647
1,302
2,985
Orientation
+
+
+
+
+
+
+
+
+
+
+
+
Predicted Product
chromosome segregation protein SMC
hypothetical protein
type I secretion outer membrane protein, TolC
family
Cation efflux system protein CusB precursor
Cation efflux system protein CusA
hypothetical protein
Cation efflux system protein CusF precursor
Lipoprotein signal peptidase
Sodium/calcium exchanger protein
Nitrogen regulatory protein P-II
Cation efflux system protein CzcA
Nickel and cobalt resistance protein CnrB
Cation efflux system protein CzcC
hypothetical protein
putative cadmium-transporting ATPase
Ferrous iron uptake protein
Copper export regulator
Glutamine-dependent NAD(+) synthetase
site-specific tyrosine recombinase XerD (TnpI)
Transposase, TnpA family (DDE domain)
58
C) ISXca3: 11,523 bp from X. campestris pv. vesicatoria str. 85-10 plasmid pXCV183 NC_007507
Locus Tag
XCVd0093
XCVd0094
XCVd0095
XCVd0096
XCVd0097
XCVd0098
XCVd0099
XCVd0100
XCVd0101
XCVd0102
XCVd0103
Element ID
ISXca_00001
ISXca_00002
ISXca_00003
ISXca_00004
ISXca_00005
ISXca_00006
ISXca_00007
ISXca_00008
ISXca_00009
ISXca_00010
ISXca_00011
Coordinates
352..1,389
1,377..2,444
2,635..2,838
2,890..3,603
3,612..6,575
6,906..8,132
8,412..9,599
9,606..9,869
9,894..10,295
10,292..10,552
10,742..11,308
Length
1,038
1,068
204
714
2,964
1,227
1,188
264
402
261
567
Orientation
+
+
+
+
+
Predicted Product
hypothetical protein
chromosome segregation protein SMC
hypothetical protein
Site-specific recombinase XerD
Transposase, TnpA family
Plasmid encoded RepA protein
UDP-N-acetylglucosamine kinase
hypothetical protein
tRNA(fMet)-specific endonuclease VapC
Virulence-associated protein
DNA-invertase hin
Table S3. Distribution, genomic coordinates, and genome context of the Mlt, TnpA,
TnpS, resolution site (res site), and TnpT homologues (>40% of identity)
over other Xanthomonas species, which does not carry the ISXax2
canonical element or relatives. The identity values were calculated based
on the canonical sequence of ISXax2 from X. citri 306 pXAC64. The
genome context is shown inside the brackets, and represents the orf
located up and downstream.
Not found
Xoo PXO99A
X. albilineans str. GPE
PC73
Xff 4834-R plc
[lysine aminomutase] –
[trna/rrna methyltransferase]
A) 2,246,792 .. 2,248,069
(87%)
[XopE3] - [C.H]
A) 29,119 .. 30,390 (98%)
[C.H] – [IS3 ssgr IS407 partial]
A) 4,233,640 .. 4,234,908
(95%)
Not found
Xoo MAFF 311018
Xoo BLS256
Not found
Mlt
Xoo KACC 10331
Genome or Plasmid
Not found
Not found
Not found
[VirG] – [IS256 partial]
B) 1,625,991 .. 1,626,503 (92%) –
Partial
[IS630 partial] – [TnpS]
A) 2,302,697 .. 2,305,654 (59%)
Not found
Not found
TnpA
[TnpA]
Not found
Not found
Not found
[arsenical membrane pump] –
A) 2,301,719 .. 2,302,666
(80%)
Not found
Not found
TnpS
Not found
Not found
Not found
[ISXoo5] – [TnpT]
A) 2,294,680 .. 2,294,784
(79%)
Not found
Not found
res site [TnpS to TnpT]
Not found
A) 4,947,724 ..
4,948,167 (84%) Partial
Not found
[ISXoo5] – [TnpT]
A)
2,293,677..2,294,663
(82%)
Not found
Not found
TnpT
59
Not found
Not found
Not found
Not found
Xcv pXCV183
Xcc Aw12879 pXcaw58
Xcc B100
Pseudoxanthomonas
spadix BD-a59
B) 123,255 .. 124,181 (98%)
[C.H] – [TnpA]
B) 119,656 .. 122,448 (41%)
[C.H] – [C.H]
[ TnpA] – [transcription factor
]
[alcohol dehydrogenases] – [TnpS]
B) 651,884 .. 653,152 (71%)
A) 636,754 .. 638,019 (71%)
[TnpA] – [TnpT]
A) 636,723 .. 633,745 (85%)
[IS3 ssgr IS407 partial ] – [type III]
B) 2,787,410 .. 2,788,048 (87%) –
Partial
[Peptidase] – [TnpS]
A) 2,759,319 .. 2,762,255 (59%)
[hypothetical] – [hypothetical]
C) 54,640 .. 57,432 (41%)
A) 2,763,259 .. 2,763,416
(79%)
[ATPase domain] – [TnpT]
A) 666,719 .. 666,827 (76%)
[TnpS] – [TnpT]
[endonuclease] – [secC metal-binding protein]
A) 666,842 .. 667,873
(86%)
[TnpS] – [hypothetical ]
A) 2,763,416 ..
2764465 (93%)
Partial
[hypothetical] – [TnpS]
[hypothetical] – [hypothetical]
A) 2,762,282.. 2,763,226
(90%)
[TnpT] - [TnpS]
A) 3,218 .. 3,790 (94%)
Partial
B) 101,026 .. 101532
(94%)
[Type III] – [TnpS]
A) 102,969 .. 103,979
(91%)
B) 2,739 .. 3,218 (95%)
[ Type III] – [TnpA]
A) 3,792 .. 3,983 (85%)
[TnpT] – [ TnpS]
A) 103,979 .. 104,170 (90%)
B) 18,384 .. 21,176 (41%)
[TnpS] –[Tn3 transposase - partial]
A) 6,161 .. 9,097 (59%)
[TnpS] – [hypothetical]
A) 3,983 .. 6,134 (95%) frameshift
[tnpT] – [tnpA]
[tnpS] – [repA]
C) 124,208 .. 127,144 (59%)
A) 104,170 .. 105,120 (97%)
A) 105,147 .. 108,086 (59%)
60
Abbreviations: H= hypothetical protein; C.H= conserved hypothetical protein
[H] – [transcription factor]
61
62
Figure S1. Xac 306 chr (An ISXax2 relative copy) left end (5’) compared to ISXax2
canonical element located on pXAC64. The extreme left region of 475bp
including the left TIR is located 335 kb away in another region of the Xac
chromosome and in an inverted orientation. The rearranged regions are flanked
by an insertion sequence from IS3 family, sub-group IS407 and
rhamnogalacturonan hydrolase B gene (rhgB). The genomic coordinates are
represented.
63
Figure S2. ISXax2 and MICs transposition mating out experiment scheme.