Do simple sequence repeats in replication, repair and ...

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Do simple sequence repeats in replication, repair and ...

Journal of Cell and Molecular Biology 7(2) & 8(1): 53-70, 2010 Research Article

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Do simple sequence repeats in replication, repair and recombination

genes of mycoplasmas provide genetic variability?

Seema TRIVEDI

Computational Biology Lab., Department of Zoology, JN Vyas University, Jodhpur

(Rajasthan), India

(author for correspondence; svtrived@hotmail.com)

Received: 25 December 2009; Accepted: 14 May 2010

Abstract

Simple sequence repeats (SSRs) or microsatellites are mono to hexa-nucleotide tandem repeats of DNA that

are ubiquitous in intergenic regions and coding regions of genomes. SSRs may be essential for any genome

as these repeats are found even in organisms like mycoplasmas (class Mollicutes) that have small genome

size. Mollicutes cause different diseases in vertebrates including humans, insects and plants. Antibiotics have

been developed against membrane proteins and replication related proteins like gyrase and topoisomerase.

However, some pathogens have developed immunity against these drugs. Mycoplasmas can evade host

immune response in which SSRs variations in membrane proteins play an important role. The present study

seeks presence of di- to penta-nucleotide repeats in genes associated with DNA replication, repair and

recombination in thirteen mycoplasma genomes. Association of SSRs with these genes may have potential to

provide variability for evading antibiotics against replication, repair and recombination proteins. In the

present study SSRs are present in few genes but among the repeats found, maximum repeats are present in

methylase, DNA polymerase, excinuclease and topoisomerase genes. Maximum number of repeat types are

dinucleotides but present only in M. pulmonis. Pentanucleotide repeats are present in three mycoplasmas but

tetranucleotide repeats are present in eight mycoplasmas.

Keywords: Microsatellites, Mycoplasma, repair, replication, simple sequence repeats

Mikoplazmanın replikasyon, tamir ve rekombinasyon genlerindeki basit dizi tekrarları

genetik çeşitlilik mi sağlıyor?

Özet

Basit dizi tekrarları (SSR) veya mikrosatelitler, genomun intergenik bölgelerinde ve kodlanan bölgelerinde

sıkça rastlanan, DNA’nın birden (mono) altıya (hekza) kadar ard arda gelen nükleotit tekrarlarıdır. Bu

tekrarlar mikoplazma (Mollicutes sınıfı) gibi küçük genom büyüklüğüne sahip bir organizmada bile

bulunduğundan, SSR’ler herhangi bir canlı için elzem olabilir. Mollicutes, insan, böcek ve bitkileri de içeren

omurgalılarda çeşitli hastalıklara yol açarlar. Zar proteinlerine ve giraz, topoizomeraz gibi replikasyonla

alakalı proteinlerine karşı antibiyotikler geliştirilmiştir. Ancak, bazı patojenler bu ilaçlara karşı bağışıklık

kazanmışlardır. Zar proteinlerindeki SSR varyasyonlarının mikoplazmaların konakçı hücrenin bağışıklık

tepkisinden kaçmalarında önemli bir rolü olabilir. Bu çalışma, onüç mikoplazma genomunda DNA

replikasyonu, tamiri ve rekombinasyonu ile ilgili genlerdeki ikiliden (di-) beşliye (penta-) kadar olan

nükleotit tekrarlarını araştırır. Bu genlerin SSR’ler ile ilişkisi, replikasyon, tamir ve rekombinasyon

proteinlerine karşı geliştirilen antibiyotiklerden kaçmaları için çeşitlilik sağlamada potansiyele sahip olabilir.

Bu çalışmada, SSR’ler birkaç gende mevcuttur, ama bulunan tekrarlar arasında en fazla tekrar metilaz, DNA

polimeraz, ekzinükleaz ve topoizomeraz genlerinde vardır. Tekrar tiplerinden en fazla sayıda bulunanlar

dinükleotitlerdir ancak sadece M. Pulmonis’de bulunur. Pentanükleotit tekrarları üç mikoplazmada, ama

tetranükleotit tekrarları sekiz mikoplazmada mevcuttur.

Anahtar Sözcükler: Mikrosatelitler, mikoplazma, tamir, replikasyon, basit dizi tekrarları


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Seema TRIVEDI

Introduction

Simple sequence repeats (SSRs) or microsatellites

are mono to hexa-nucleotide tandem repeats of

DNA that are ubiquitous in all genomes studied

so far. SSRs are present not only in intergenic

regions of a genome, but may also be found in

introns and exons of coding regions (Karlin et al.,

1997; Bachtrog et al., 1999; Bachtrog et al., 2000;

Butcher et al., 2000; Chambers and MacAvoy,

2000; Toth et al., 2000). It is possible that SSRs

are essential part of any genome as these repeats

are present even in organisms like mycoplasmas

that have small genome size (Hancock, 1996a).

These repetitive DNA may be involved in

different functions like chromatin organization,

gene regulation, evading host-immune responses,

recom bination hotspots and facilitating genome

rearrangements, affecting protein structure thus

possibly protein-protein interactions etc. (Mrázek,

2006). Interestingly, it has also been found that

SSRs may provide variability to host for evading

pathogens. This is reported in case of house

finches (Carpodacus mexicanus) that show

multilocus heterozygosity that could result in

reduced susceptibility to Mycoplasma

gallisepticum infection (Hawley et al., 2005).

Similarly, Porcine C3 gene (high homology with

human C3) involved in phagocytosis, inflamema

tion and immunoregulation to destroy infectious

micro-organisms, possess Tn SSR in the

3’flanking region which may be helpful in

resisting infections (Mekchay et al., 2003).

Mycoplasmas (class Mollicutes) are parasites

or commensals that may cause different diseases

in vertebrates including humans, insects and

plants. Repetitive sequences like RepMp and

MgPar elements are involved in homologous

recombination of parts of P1, P40, P90 and P110

proteins of Mycoplasma pneumoniae and Myco

plasma genitalium. Recombination events

mediated with help of MPN490- and MG339encoded

proteins (RecA homo logs) in genes of

immunogenic adhesion proteins result in

variations that may play eminent roles in immune

evasive strategies (Sluijter et al., 2009). Repeated

sequences, DR-1 and DR-2, within the putative

cytadhesin pvpA gene of M. gallisepticum are

present in isolates from Chinese poultry farms.

Approximately 30 or more proline residue repeats

and 7-10 repeats of the tetrapeptide motif may

affect functionality of PvpA as an adhesin molecule

(Jiang et al., 2009). Similarly, insertion sequences

(IS3, IS4 and IS30) in Mycoplasma bovis

(Lysnyansky et al., 2009) and variable-number

tandem-repeats (VNTRs) associated with coding

sequences that can provide genetic diversity are

present in Mycoplasma hyopneumoniae strains,

Mycoplasma agalactiae type strain PG2,

Mycoplasma mycoides subspecies mycoides. These

IS and VNTRs possibly code for amino acid repeats

and can affect cell adhesion and interactions with the

host immune system (de Castro et al., 2006;

McAuliffe et al., 2007; McAuliffe et al., 2008).

Tandem amino acid repeats in M. pneumoniae

RepMP1-containing genes and Myco plasma

pulmonis Vsa proteins may have regulatory functions

(Simmons and Dybvig, 2007; Musatovova et al.,

2008). Amino acid repeats that may affect antigenic

response are present in haemagglutinins (immuno

genic, variably expressed, surface proteins) of M.

synoviae (VlhA) (Bencina, 2002).

Though SSRs have been investigated in

mycoplasmas, the focus of studies has not been

replication, repair and recombination genes. This

study was undertaken to seek answer to questions

like whether replication, repair and recombination

genes in mycoplasmas are associated with SSRs.

How can SSRs association with these genes be

beneficial to these organisms? The present study on

SSRs in replication, repair and recombination genes

in thirteen mycoplasmas may indicate mutational

hotspots that may help these organisms evade

antibiotics against these genes. On the other hand,

these mutational hotspots may prove to be target sites

for developing drugs to prevent such evasive

strategies of the organisms.

Method

Obtaining sequences and genome information

Replication, repair and recombination related gene

sequences (total numbers given in Table-1) of

thirteen myco plasmas (Mycoplasma arthritidis

158L3-1, Mycoplasma capricolum subsp capricolum

California kid ATCC 27343, Mycoplasma

gallisepticum strain R, Mycoplasma genitalium G-37,

Mycoplasma hyopneumoniae 232, Mycoplasma

hyopneumoniae 7448, Mycoplasma hyopneumoniae

J, Mycoplasma mobile 163K, Mycoplasma mycoides

SC PG1, Mycoplasma penetrans HF-2, Mycoplasma

pneumoniae M129, Mycoplasma pulmonis UAB


CTIP and Mycoplasma synoviae 53) were

downloaded from “The Comprehensive Microbial

Resource” (CMR http://cmr.jcvi.org/cgi-bin/

CMR/CmrHomePage.cgi). Total genome length,

CG richness, coding percentage and number of

Table 1. List of mycoplasmas and genome details

Organism

Genome

length

nt

Genome

CG%

Coding

%

SSRs in Mycoplasma replication genes

protein coding genes for each Mycoplasma were

downloaded from NCBI (http://www.ncbi.nlM.

nih.gov/sites/entrez?db=genome&cmd=Retrieve&do

pt=Overview&list_uids=) (Table 1).

Total

protein

genes

Total

replication

and repair

genes

% of

genes

with

repeats

Mycoplasma

arthritidis 158L3-1 820453 30 88 631 32 3.13

Mycoplasma

capricolum subsp

capricolum California

kid ATCC 27343

Mycoplasma

1010023 23 88 812 45 8.89

gallisepticum strain R

Mycoplasma

996422 31 87 726 30 3.33

genitalium G-37

Mycoplasma

580076 31 90 475 28 3.57

hyopneumoniae 232

Mycoplasma

892758 28 89 691 34 2.94

hyopneumoniae 7448

Mycoplasma

920079 28 85 657 35 2.86

hyopneumoniae J

Mycoplasma mobile

897405 28 87 657 34 2.94

163K

Mycoplasma mycoides

777079 24 90 633 39 5.13

SC PG1

Mycoplasma penetrans

1211703 23 81 1016 51 1.96

HF-2

Mycoplasma

1358633 25 88 1037 76 2.63

pneumoniae M129

Mycoplasma pulmonis

816394 40 87 689 36 5.56

UAB CTIP 963879 26 89 782 53 9.43

Mycoplasma synoviae

53 799476 28 88 659 36 8.33

55


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Seema TRIVEDI

SSR search

Replication, repair and recombination related

gene sequences of all thirteen mycoplasmas were

subjected to repeat search programme SPUTNIK

(http://espressosoftware.com/sputnik/index.html).

SPUTNIK looks for di-, tri-, tetra- and pentanucleotide

repeats; tolerates insertions, mismatch

es and deletions but these affect the overall score.

Repeats found through this search were not

grouped i.e. the 5’ to 3’ or vice-a versa and the

complements were not put together. For example,

dinucleotide repeat CA was not grouped with AC,

TG or GT. Hence, each repeat was treated as

separate motif.

SSR CG richness and length

Length of an SSR was measured in nucleotides

(base pairs). However, repeat length given as

output from SPUTNIK was adjusted to nearest

divisible value i.e. dinucleotide repeat length

should be divisible by 2, trinucleotide by 3,

tetranucleotide repeat by 4 and pentanucleotide

repeat by 5. For example, if repeat length for

dinucleotide repeats was indicated as 11nt by

SPUTNIK output; this was adjusted to 10nt, for

trinucleotide repeats if the length was 14nt it was

adjusted to 15nt or if it was 16nt or 17nt it was

considered as 15nt.

Statistical analysis

For statistical analysis, SPSS (Version 16.0) was

used. One way ANOVA followed by Tukey’s

HSD at 95% confidence level was done to seek

significant difference in between the thirteen

species repeat number as well different replica

tion, repair and recombination genes.

Pearson’s ‘r’ two tailed correlation analysis of

genome size and CG richness, sequence lengths

of replication, repair and recombination genes and

CG richness vis-à-vis repeat numbers, length and

.

CG richness of total repeats and repeat types was

done.

Results

Total SSRs, repeat types and motifs in thirteen

mycoplasmas replication, repair and recombination

genes

Figure 1 shows total numbers of SSRs and repeat

types present in replication, repair and recombination

genes in genomes of thirteen mycoplasmas where M.

pulmonis has the maximum number of repeats

followed by M. capricolum and M. synoviae (5, 4

and 3 respectively).

Among repeat types, dinucleotides (five) are

present only in M. pulmonis. Maximum numbers of

tri nucleotides are in M. capricolum. Presence of

other repeat types in replication, repair and

recombination genes of mycoplasmas are given in

Figure 1.

There is diversity in repeat motifs found in the

present study among which maximum frequency

(four) is of tetranucleotide repeat ATTT followed by

dinucleotide repeat motifs AG (Table-2).

Total replication, repair and recombination genes

associated with SSRs in mycoplasmas

Methylase genes have maximum number of total

repeats (eight) followed by DNA polymerase,

excinuclease and topoisomerase genes (three in each)

among replication, repair and recombination genes in

thirteen mycoplasmas (Figure 2). Among repeat

types total dinucleotide followed by tetra nucleotide

repeats as well as the motifs AG and ATTT have

maximum frequency (all in methylase genes)

(Figures 2 and 3).

Comparison of total repeats and repeat types in

replication, repair and recombination genes of each

Mycoplasma is given in Figure 4 and motifs in Table

3, where once again methylase genes in M. pulmonis

have maximum repeats.


SSRs in Mycoplasma replication genes

Figure 1. Total SSRs and SSR types in thirteen mycoplasmas replication, repair and recombination genes.

Di: Dinucleotides, Tri: Trinucleotides, Tetra: Tetranucleotides, Penta: Pentanucleotides, Total: Total Repeats.

Figure 2. Total SSRs and SSR types in total replication, repair and recombination genes of mycoplasmas. Di:

Dinucleotides, Tri: Trinucleotides, Tetra: Tetranucleotides, Penta: Pentanucleotides, Total: Total Repeats.

57


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Seema TRIVEDI

Table 2. SSR motifs and motif numbers in total replication, repair and recombination genes of thirteen

mycoplasmas. Di: Dinucleotide, Tri: Trinucleotide, Tetra: Tetranucleotide, Penta: Pentanucleotide.

Repeat

Name

Di

Tri

Tetra

Penta

Length of repeats

Organism Motifs

AG CA GA

M. pulmonis 3 1 1

Grand Total 3 1 1

AAG AAT AGA GAA TGA TGT

M. arthritidis 1

M. capricolum 1 1 1

M. mobile 1

M. penetrans 1

M. synoviae 1

Grand Total 1 2 1 1 1 1

AATT ATTT TTTA TTTC

M. capricolum 1

M. genitalium 1

M. hyopneumoniae 232 1

M. hyopneumoniae 7448 1

M. hyopneumoniae J 1

M. mobile 1

M. mycoides 1

M. penetrans 1

Grand Total 1 4 2 1

AAAAT AAAGC AATTG ACCAA CAAAC

M. gallisepticum strain R 1

M. pneumoniae 1 1

M. synoviae 1 1

Grand Total 1 1 1 1 1

Repeats in thirteen mycoplasmas are not very

long as maximum repeat lengths are 28nt and

26nt which are in methylase genes (dinucleotide

CA and GA respectively in M. pulmonis)

followed by 24nt which are in methylase and

gyrase genes (dinucleotide AG in M. pulmonis

and trinucleotide AAG in M. arthritidis

respectively) (Figure 5 and Table 4).

CG richness of SSRs

There is variation (0% to 50%) in CG richness of

SSRs in replication, repair and recombination genes

of mycoplasmas (Table 5). CG% differs in the same

gene group among different Mycoplasma. For

example, DNA polymerase shows different CG

richness in different Mycoplasma (0% in M.

synoviae, 33.33% in M. mobile and 40% in M.

pneumoniae).


SSRs in Mycoplasma replication genes

Figure 3. SSR motifs in total replication, repair and recombination genes in mycoplasmas. legends represent

the motifs as per the bar chart.

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Seema TRIVEDI

Figure 4. Total SSRs and SSR Types In Replication, Repair and Recombination Genes In Thirteen Mycoplasmas. Di: Dinucleotides, Tri:

Trinucleotides, Tetra: Tetranucleotides, Penta: Pentanucleotides, Total: Total Repeats.


SSRs in Mycoplasma replication genes

Table 3. Number of SSR motifs in replication, repair and recombination genes of thirteen

mycoplasmas.

Organism Motif

Conserved

Hypothetical Protein

DNA Polymerase

Endonuclease

Excinuclease

M. arthritidis AAG 1

Gyrase

Ligase

Methylase

Methyltransferase

P35 Lipoprotein

Homolog

AGA 1

M. capricolum

TGA

TGT 1

1

TTTA 1

M. gallisepticum ACCAA 1

M. genitalium AATT 1

M. hyopneumoniae 232 ATTT 1

M. hyopneumoniae 7448 ATTT 1

M. hyopneumoniae J ATTT 1

M. mobile

GAA

ATTT

1

1

M. mycoides TTTA 1

M. penetrans

AAT

TTTC 1

1

M. pneumoniae

AAAGC

CAAAC 1

1

AG 3

M. pulmonis

CA 1

GA 1

AAT 1

M. synoviae

AAAAT 1

AATTG 1

Replication Initiator

SSBP

Topoisomerase

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Seema TRIVEDI

Figure 5. Lengths of total SSRs and SSR type in total replication, repair and recombination genes of mycoplasmas. SSR lengths in

number of nucleotides (nt).


SSRs in Mycoplasma replication genes

Table 4. Repeat lengths (nt) in total SSR types and SSR motifs in replication, repair and recombination genes

of thirteen mycoplasmas. Di: Dinucleotides, Tri: Trinucleotides, Tetra: Tetranucleotides, Penta:

Pentanucleotides, Total: Total Repeats.

.

Gene Organism SSR Motif

SSR

Length

Conserved Hypothetical Protein M. synoviae Penta AATTG 10

DNA Polymerase

M. mobile Tri GAA 12

M. pneumoniae Penta CAAAC 10

M. synoviae Tri AAT 12

Endonuclease M. penetrans Tetra TTTC 12

Excinuclease

M. capricolum Tri AGA 12

M. pneumoniae Penta AAAGC 15

M. synoviae Penta AAAAT 10

Gyrase M. arthritidis Tri AAG 24

Ligase M. genitalium Tetra AATT 12

Methylase

M. hyopneumoniae 232 Tetra ATTT 12

M. hyopneumoniae 7448 Tetra ATTT 12

M. hyopneumoniae J Tetra ATTT 12

M. pulmonis Di GA 26

CA 28

14

AG 16

24

Methyltransferase M. mobile Tetra ATTT 12

P35 Lipoprotein Homolog M. penetrans Tri AAT 18

Replication Initiator M. capricolum Tri TGT 12

SSBP M. capricolum Tri TGA 12

Topoisomerase

M. capricolum Tetra TTTA 12

M. gallisepticum Penta ACCAA 10

M. mycoides Tetra TTTA 12

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Seema TRIVEDI

Table 5. CG richness of SSRs and motifs in replication, repair and recombination genes in thirteen

mycoplasmas. Di: Dinucleotide, Tri: Trinucleotide, Tetra: Tetranucleotide, Penta: Pentanucleotide

SSR

CG%

0

Gene Name Repeat Motif Organism

SSR

Number

DNA Polymerase Tri AAT M. synoviae 53 1

Excinuclease Penta AAAAT M. synoviae 53 1

Ligase Tetra AATT M. genitalium G-37 1

Methylase Tetra ATTT M. hyopneumoniae 232 1

M. hyopneumoniae 7448 1

M. hyopneumoniae J 1

Methyltransferase Tetra ATTT M. mobile 163K 1

P35 Lipoprotein

Homolog

Tri AAT

M. penetrans HF-2 1

Topoisomerase Tetra TTTA M. capricolum 1

M. mycoides 1

20 Conserved

Hypothetical Protein

Penta AATTG

M. synoviae 53 1

25 Endonuclease Tetra TTTC M. penetrans HF-2 1

33.33 DNA Polymerase Tri GAA M. mobile 163K 1

Excinuclease Tri AGA M. capricolum 1

Gyrase Tri AAG M. arthritidis 158L3-1 1

Replication Initiator Tri TGT M. capricolum 1

40

SSBP Tri TGA M. capricolum 1

DNA Polymerase Penta CAAAC M. pneumoniae M129 1

Excinuclease Penta AAAGC M. pneumoniae M129 1

Topoisomerase Penta ACCAA M. gallisepticum strain R 1

50 Methylase Di AG M. pulmonis 3

CA M. pulmonis 1

Discussion

SSRs in genomes of virus, prokaryotes and

eukaryotes are present not only in intergenic

regions but also in introns and exons of coding

sequences (Hancock, 1996a and b; Karlin et al.,

1997; Bachtrog et al., 1999; Bachtrog et al., 2000;

Butcher et al., 2000; Toth et al., 2000; Trivedi,

2003, 2004, 2006; Mrázek et al., 2007). Studies in

different genomes have also shown associations

GA M. pulmonis 1

of SSRs with replication, repair and recombination

genes, housekeeping genes or membrane proteins

(Trivedi, 2003; Mrázek et al., 2007; Guo and

Mrázek, 2008). Antigenic variations facilitated by

SSRs in host-adapted pathogens like mycoplasmas

have been reported (Guo and Mrázek, 2008).

However, studies have so far not focused on


association of SSRs with replication, repair and

recombination genes.

Total repeats

Mycoplasmas have small genomes possibly due

to genome reduction. It is possible that some

SSRs may have played a role in Mycoplasma

evolution (Hancock, 1996a; Rocha and

Blanchard, 2002) which could be due to

mutational bias towards SSR reduction instead of

expansion if not maintained by selection (Metzgar

et al., 2002).

The present study shows few repeats in total

replication, repair and recombination genes which

corresponds to studies in whole genome studies

on mycoplasmas (Mrázek et al., 2007) but with

some differences from the reported number of

repeats in these genes in M. hyopneumoniae (Guo

and Mrázek, 2008). This difference is possibly

due to difference in the algorithms used in the

present study and the analysis criteria. ANOVA

does not show any significant difference between

the thirteen mycoplasmas nor between genes of

each Mycoplasma in the present study. Besides

this, present study does not show any correlation

of genome length with any parameter as indicated

in the material and method section. This is

consistent with some studies (Hancock, 2002;

Lim et al., 2004) but contrary to studies done

earlier in eukaryotes and prokaryotes (Hancock,

1996b; Primmer et al., 1997; Achaz et al., 2002;

Trivedi, 2004 and 2006; Mrázek et al., 2007).

Though the focus of present study is on specific

genes, this lack of correlation of genome size may

indicate confirmation of the fact that genome size

is not expanding and possibly SSRs (in total

genomes) that are so few in number are playing a

role in genome reduction in Mycoplasma

(Hancock 1996a; Mrázek et al., 2007; Guo and

Mrázek, 2008).

Genome CG% show significant positive

correlation only with penta nucleotide number

and length. Similarly, significant positive

correlation of average CG% of total replication,

repair and recombination genes with pentanucleo

tide lengths (P< 0.05, 0.587*) is seen in the

present study. There is significant positive

correlation (P< 0.01, 0.931**) of total SSR

number with percentage of total replication, repair

and recombination genes with repeats. Total SSR

numbers show significant positive correlation

with total dinucleotide number (P< 0.01, 0.700**)

and total tetranucleotides show significant

SSRs in Mycoplasma replication genes

negative correlation with pentanucleotide number

(P< 0.05, -0.659*).

This data is not sufficient to draw conclusion that

positive correlation of percentage of genes having

repeats with total repeats may be an indication of

probable increase in repeat number in these genes in

other pathogens as well. It would be interesting to

investigate repeats in those micro-organisms as well

against which antibiotics are being used. Antibiotic

treatment would alter environment and these

pathogens may develop different strategies to evade

these challenges. These evasive strategies may

involve the membrane proteins or replication, repair

and recombination related genes or gene products

depending on the target proteins of antibiotics.

SSR average length

Many organisms studied so far have shown

differences in not only abundance of SSRs but also in

tolerance for length of SSRs. For example, archaea

like Methanosarcina mazei strain Goe1,

Methanobacterium thermoautotrophicum DH,

Helicobacter pylori have some SSRs that are long

compared to other species (Field and Wills, 1998;

Trivedi, 2006). Among vertebrates, cold-blooded

vertebrates like turtles have long repeats (Chambers

and MacAvoy, 2000); whereas humans have longer

SSRs compared to homologues in chimps (Cooper et

al., 1998).

Long SSRs have not been found in the present

study including other studies in M. penetrans, M.

mobile, and M. synoviae. Short A(n) and T(n) repeats

are abundant in M. hyopneumoniae and M. pulmonis

(Mrázek, 2006). The present study shows significant

positive correlation between total SSR average

length with dinucleotide number and length as well

as tetranucleotide CG% (P< 0.05, 0.563*, 0.563* and

P< 0.01, 1.000** respectively). Dinucleotide average

length has significant positive correlation with total

SSR as well as dinucleotide numbers and CG% of

total SSRs (P< 0.01, 0.700**, 1.000** and 0.870**

respectively). Significant positive correlation of

trinucleotide length is seen with trinucleotide number

but negative correlation with pentanucleotide CG%

(P< 0.05, 0.672* and P< 0.01,-1.000** respectively).

Tetranucleotide average length has positive

correlation with tetranucleotide number but negative

correlation with pentanucleotide number as well as

length (P< 0.01, 1.000**, P< 0.05, -0.659* and P<

0.01, -0.688** respectively). There is positive

correlation of pentanucleotide average length with

pentanucleotide number (P< 0.01, 0.963**).

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Seema TRIVEDI

Reports have suggested that in prokaryote

genomes, significantly long SSRs are generally

rare but association of long mono-, di-, tri-, and

tetranucleotides are mostly restricted to hostadapted

pathogens. Besides this, a number of long

SSRs are also associated with housekeeping

genes, including rRNA and tRNA genes, genes

encoding ribosomal proteins, amino acyl-tRNA

synthetases, chaperones, and important metabolic

enzymes. Statistically significant associa tions

between SSRs and gene functional classifications

suggest that most long SSRs are not related to a

particular cellular function or process (Guo and

Mrázek, 2008). This conclusion may be true for

many organisms and particularly prokaryotes, but

pilot study on silkworm genes has indicated

biased association of SSRs with genes associated

with replication and repair process though length

of SSRs was not focus of the study (Trivedi,

2003). In Mycobacterium leprae long SSRs are

mostly associated with pseudogenes and may be

contributing to gene loss following the adaptation

to an obligate pathogenic lifestyle. The authors

speculate that LSSRs may have played a similar

role in genome reduction of other host-adapted

pathogens (Guo and Mrázek, 2008). Similarly in

M. gallisepticum SSRs are biased towards

deletion unlike eukaryotic genomes where they

are biased towards expansion (Metzgar et al.,

2002).

Among prokaryotes, it is proposed that the

differences could be due to functional nature of

these repeats or because some of these genomes

lacks mismatch repair (Field and Wills, 1998).

However, the latter reason does not apply to

eukaryotes and particularly to differentces

between chimpanzee and human SSRs.

SSR average CG%

Total SSRs CG% show positive correlation with

total SSR and dinucleotide number (P


epeats. Frame-shift mutations caused in either di-

or tetra-nucleotide (of the present study) repeats

in these genes may facilitate altered methylation

patterns and influence gene expression thus may

indirectly contribute to antigenic variations that

could help in evasion of host immune response

(Rocha and Blanchard, 2002; Mrázek, 2006; Guo

and Mrázek, 2008).

DNA polymerase, excinuclease, gyrase and

topoisomerase

Among replication, repair and recom bination

genes; M. mobile, M. pneumoniae, M. synoviae

have repeats in DNA polymerase. Excinuclease

genes have SSRs in M. capricolum, M.

pneumoniae and M. synoviae and topoisomerase

genes have SSRs in M. capricolum, M.

gallisepticum and M. mycoides. However, gyrase

genes have SSR only in M. arthritidis 158L3-1

and single strand binding proteins (SSBP) only in

M. capricolum. SSRs association with DNA

polymerase, gyrase and SSBP genes besides other

essential as well as housekeeping genes has also

been reported in other prokaryotes including

mycoplasmas. Guo and Mrázek (2008) speculate

that variation in essential genes like replication,

repair and recombination genes (except

methylase) in prokaryotes may not be helpful but

possibly these SSRs affect transcription initiation.

However, there is a possibility that these SSRs

are once again providing most of these pathogens

opportunity to evade antibiotics. This hypothesis

gets its support from the fact that many

prokaryotes including mycoplasmas are becoming

resistant to fluoroquinolones that are broadspectrum

antibiotics like ofloxacin (OFX),

ciprofloxacin (CFX) and sparfloxacin (SFX)

targeted against proteins like topoisomerase II

family, DNA gyrase and topoisomerase IV. Some

mycoplasmas have developed resistance against

these drugs due to mutations in target regions of

these enzymes (Bébéar et al., 1998). Therefore it

is suggested that those drugs would be more

effective that would target at least two

proteins/genes simultaneously to block of slow

down DNA replication. This is because two

mutations simultaneously in two genes to evade

antibiotics would be rare and hence it may be

more effective as it has been suggested in studies

done on Staphylococcus aureus (Fournier et al.,

2000).

Similarly, there are mutants against antibiotics

that target Mycoplasma ribosomes (Pereyre et al.,

SSRs in Mycoplasma replication genes

2002). It is possible that SSRs in the rRNA genes

(Guo and Mrázek, 2008) (but not part of this study)

may be providing genetic variability to the pathogen

to evade altered living environment due to antibiotics

against their ribosomes.

Functions of SSRs

SSRs may play diverse roles in genomes of different

organisms. Some of these repeats act as contingency

loci in association with families of surface antigens

in pathogens, affect DNA supercoiling, gene

expression or recombination hotspots. M.

gallisepticum major surface protein pMGA

expression switching from pMGA1.1 to pMGA1.2 is

associated with (GAA)(12) repeat and variations in

length (Glew et al., 2000; Liu et al., 2000; Liu et al.,

2002). M. pneumoniae, M. genitalium, Ureaplasma

urealyticum and M. pulmonis have recombination

potentials in different genomic regions. In particular

M. pulmonis has illegitimate recombination at the vsa

locus. M. pneumoniae and M. genitalium adhesins

have large distant repeats that may be responsible for

homologous recombination for variation (Rocha and

Blanchard, 2002). However, M. hyopneumoniae have

fewer repeats compared to other mycoplasma.

Therefore, it is intriguing how M. hyopneumoniae

evades host immune response and establishes chronic

infection in absence of repeats (Minion et al., 2004).

Not all SSRs may be related with these functions.

For example, repeats in M. hyopneumoniae may not

be contingency loci as these appear independent of

location upstream or downstream of genes. Further,

among 3 M. hyopneumoniae strains study shows that

the An and Tn repeats may be rarely involved in

genome rearrangements (Mrázek, 2006).

Conclusion

The diversity of repeat numbers and motifs may be

due to diversity of host environments and selection

pressures that different mycoplasmas live in. It is

also possible that SSRs are present in regions of

those segments where the amino acid coded by SSRs

may help in flexibility of protein fold (disordered

region). This may enable the protein to be more

flexible in terms of protein-protein interactions and

may have multiple partners, hence making them

possible hubs of protein interaction networks

(Mrázek, 2006). However, in the present study, the

possible role of SSRs in these genes/proteins

involves DNA binding capacity. Any mutation in

these SSRs or other regions of the protein may affect

67


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Seema TRIVEDI

DNA-protein interaction and hence affect

replication efficiency. Since there are antibiotics

targeted against replication enzymes, these SSRs

may be involved in providing platform for small

mutations that are not large enough to totally

affect functions of DNA replication, repair and

recombination but are efficient enough to evade

the antibiotics.

Acknowledgements

The author is grateful to Department of

Biotechnology (DBT, Ministry of Science and

Technology, India) for funding this work.

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