TAT-Pathway-Dependent Lipoproteins as a Niche-BasedAdaptation in Prokaryotes

Hamsanathan Shruthi • Mohan Madan Babu •

Krishnan Sankaran

Received: 1 October 2009 / Accepted: 3 March 2010

� Springer Science+Business Media, LLC 2010

Abstract Bacterial lipoproteins, characterized by the

N-terminal N-acyl S-diacylglyceryl Cysteine, are key

membrane proteins in bacterial homeostasis. It is generally

thought that during the modification lipoprotein precursors

are translocated via the Sec-machinery in an unfolded state.

The recent discovery of twin-arginine translocation (TAT)

machinery, meant for exporting folded-proteins, and the

presence of TAT-type signal sequences in co-factor-con-

taining (hence already folded) lipoproteins, prompted us to

investigate its role and significance in lipoprotein biosyn-

thesis. We systematically analyzed 696 prokaryotic gen-

omes using an algorithm based on DOLOP and TatP rules

to predict TAT-pathway-dependent lipoprotein substrates.

Occurrence of the deduced TAT-pathway-dependent lipo-

protein substrates in relation to genome size, presence or

absence of TAT machinery, and extent of its usage for

lipoprotein export and habitat types revealed that unlike the

host-obligates, the free-living prokaryotes in complex

hostile environments (e.g., soil) depend more on TAT-

exported lipoproteins. Functional classification of the pre-

dicted TAT-dependent lipoproteins revealed enrichment in

hydrolases and oxido-reductases, which are fast-folding

and co-factor-containing proteins. The role of the TAT

pathway in the export of folded-lipoproteins and in

niche-specific adaptation for survival has important impli-

cations not only in lipoprotein biosynthesis, but also for

protein and metabolic engineering applications.

Keywords TAT-box � Lipobox � Co-factor containing

lipoproteins � DOLOP � TatP � Habitat � Oxido-reductases �Hydrolases � Binding proteins

Introduction

Bacteria need to transport proteins across their membrane

for constant interaction with their extracellular milieu. The

general secretory pathway, referred to as Sec-pathway, has

been known to transport the majority of proteins across the

cytoplasmic membrane. Proteins exported through this

pathway possess a characteristic N-terminal extension that

is cleaved off after the translocation by the transmembrane

signal peptidases (Spases I&II) on the periplasmic side of

the membrane (Pugsley 1993). Signal peptidase II is spe-

cific to prolipoproteins, whose signal sequence contains the

lipobox, [LVI][ASTVI][GAS]C at its C-terminus. In the

lipoprotein biosynthesis, the invariant Cys gets lipid

modified and only then is the signal peptide cleaved off and

the partially modified N-terminal Cys is fatty acylated. The

acyl moieties anchor the lipoproteins to either the inner or

outer membrane (Sankaran and Wu 1994).

The Sec apparatus has been demonstrated to transport

proteins in the unfolded state (Liu et al. 1989). Such a

system is not suited to transport fully folded proteins, but a

machinery for exporting such proteins is available via the

Twin Arginine Translocase pathway (TAT) (Lee et al.

2006). This pathway exports fully folded proteins con-

taining a signal sequence with a characteristic semi-con-

sensus sequence (TAT-box), S/T-R-R-F-L-K, at the

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00239-010-9334-2) contains supplementarymaterial, which is available to authorized users.

H. Shruthi � K. Sankaran (&)

Centre for Biotechnology, Anna University,

Chennai 600 025, Tamil Nadu, India

e-mail: ksankran@yahoo.com; ksankaran@annauniv.edu

M. Madan Babu

MRC-Laboratory of Molecular Biology, Hills Road,

Cambridge CB2 0QH, UK

123

J Mol Evol

DOI 10.1007/s00239-010-9334-2

boundary of the n-region and the h-region; the twin argi-

nines are almost invariant and a signature for identifying

TAT substrates (Dilks et al. 2003). TAT as an export

system has been studied extensively using the prototypical

signal sequence of E. coli trimethylamine oxido-reductase

(TorA) (Lee et al. 2006). Other TAT-signal sequences,

DmsA, SufI FdnG, FdoG, HyaA, from E. coli (DeLisa et al.

2003), PhoD from Bacillus subtilis (Jongbloed et al. 2002)

and PhoA, PhoC from Streptomyces coelicolor (Apel et al.

2007) have also been used. Heterologous proteins such as

the green fluorescent protein, human tissue plasminogen

activator, and murine scFv and Fab antibody fragments

were found to require the TAT pathway for the export of

functional protein to the periplasm (Lee et al. 2006;

Ribnicky et al. 2007).

Although experiments, as well as bioinformatic analy-

ses, highlight the importance of the TAT machinery in the

translocation of folded proteins, its role in prolipoprotein

translocation (Hutchings et al. 2009) has not been ade-

quately probed. Presence of a lipobox sequence along with

the TAT motif in the protein sequences of Streptomyces

coelicolor, Legionella pneumophila, and Haloferax volca-

nii, suggests the existence of TAT-dependent lipoproteins

(De Buck et al. 2004; Dilks et al. 2005; Gimenaz et al.

2007). Employing TAT mutants of S. coelicolor, putative

TAT lipoprotein substrates, peptidylprolyl cis–trans isom-

erase, a putative sugar-binding protein, an iron–sulfur

binding protein and a putative secretory protein were

shown to be TAT-dependent (Widdick et al. 2006). Site-

directed mutagenesis of TAT signals of the iron-binding

protein, DsbA-like thioredoxin domain protein, and malt-

ose binding protein in H. volcanii, resulted in their accu-

mulation in the cytoplasm. Further, it was shown that the

lipoprotein signal peptidase inhibitor, Globomycin, inhib-

ited the maturation of these putative TAT substrates

(Gimenaz et al. 2007). Curiously, it was observed that the

TAT substrate, [NiFeSe] hydrogenase (HysAB) of Des-

ulfovibrio vulgaris Hildenborough has the TAT-box in the

signal sequence of the small subunit and the Lipobox in the

N-terminal region of the large subunit. Mass-spectrometric

data supporting lipid modification of this protein were

reported recently (Valente et al. 2007).

The prediction tools for identifying either putative TAT

substrates or bacterial prolipoproteins are well established.

Among these, the available TAT prediction tools are

trained only to recognize the TAT motif and an uncharged

stretch of at least 13 amino acids downstream, as in Tat-

FIND or TAT motif with Spase-I cleavage region as in

TatP (Bendtsen et al. 2005; Dilks et al. 2003). Bacterial

lipoprotein prediction tools do not distinguish between Sec

or TAT signals (Babu et al. 2006). These tools are obvi-

ously not suitable to predict potential TAT-dependent

lipoproteins. Hence a new program was written combining

the bacterial lipoprotein prediction tool that was published

recently (available in the bacterial lipoprotein web site,

DOLOP; Babu et al. 2006) and the TAT substrate predic-

tion (TatP) tool described by Bendtsen et al. (2005). Using

this new program, we have analyzed a large number of

prolipoprotein sequences from over 600 prokaryotic gen-

omes for the presence of TAT signals to obtain insight into

their occurrence and the purpose for their presence.

Methods

The completed genome sequences were obtained from the

ftp site of NCBI (ftp.ncbi.nih.gov). TAT-dependent lipo-

protein sequences were extracted using an in-house PERL

script, ‘‘TAT LIPO.’’ The program was developed by

combining DOLOP lipoprotein identification with TatP

server algorithm (Bendtsen et al. 2005; Babu et al. 2006)

.The script was trained to search for

• Presence of [LVI][ASTVI][GAS]C within the first 50

amino acids of the sequence starting with methionine.

• The sequence should contain a positively charged

residue within the first seven amino acids.

• The sequence should have an uncharged amino acid

stretch with a minimum of seven amino acids.

The sequences satisfying these conditions were taken as

lipoproteins. Among the predicted lipoproteins, the con-

sensus pattern, RR.[FGAVML][LITMVF], which is char-

acteristic of TAT dependency, was identified to predict

TAT-dependent lipoproteins (Bendtsen et al. 2005).

TAT-dependent lipoproteins were identified from 696

prokaryotic genomes and categorized based on their func-

tion. Information about the lifestyle for these organisms

was obtained from the NCBI microbial genomes website

(http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). The func-

tional classification was carried out by grouping the pre-

dicted TAT-dependent lipoproteins and providing a keyword

search for enzymes, transporters/binding proteins, and

hypothetical proteins.

The essential TAT components required for functional

TAT machinery are TatA/E and TatC. The presence or

absence of these components in 696 genomes was analyzed

by PSI-BLAST and its iterations (Dilks et al. 2003).

Functional Assignment to Hypothetical

TAT-Dependent Lipoproteins

FASTA sequences of 647 hypothetical proteins among the

predicted TAT-lipoproteins were extracted from protein

database of NCBI (http://www.ncbi.nlm.nih.gov/sites/entrez?

db=protein&cmd=search&term=) by providing the corre-

sponding accession ID. The SCOP domains to the FASTA

J Mol Evol

123

sequences were assigned using the SUPERFAMILY Hidden

Markov Models that is made available through the sequence

search facility in the SUPERFAMILY database (www.

supfam.org) (Wilson et al. 2009). The query sequences that

had significant e-value scores (\10-3) were retrieved (Gough

et al. 2001) and were further classified based on their func-

tions as described above.

Results

The New Algorithm Identifies TAT-Dependent

Lipoproteins Reliably and Reveals the Absence

of Correlation with Genome Size and Total Number

of Lipoproteins

Since there are not many experimentally verified TAT-

dependent lipoproteins, the accuracy and reliability of the

algorithm were tested on the basis of the occurrence of

essential components of the TAT pathway, TatA/E, and

TatC, as done by Dilks et al. (2003) for TatFIND. Among

the 696 bacterial and archaeal genomes analyzed, (i) 391

genomes had detectable orthologs of the TAT components

and several predicted TAT-dependent lipoproteins, (ii) 34

genomes contained no detectable orthologs of the TAT

components, but each genome did contain one or two

predicted TAT-dependent lipoproteins, 60% of which were

hypothetical proteins, (iii) 127 genomes had only the TAT

components without any predicted TAT-dependent lipo-

proteins, and (iv) 144 genomes were negative for both (See

Supplementary Table 1). It was earlier reported with a

limited dataset of prokaryotic genomes that those bacteria

lacking TatC, an essential TAT component, had small

genomes of the size 1–2 Mb (Wu et al. 2000), indicating

possible correlation between use of TAT and genome size.

As can be seen in Fig. 1a, the genome sizes of 696 pro-

karyotes fell into two groups: 368 of the genomes below

(small genomes) and 328 of the genomes above 3.5 Mb

(big genomes) (Fig. 1a). The organisms were further cat-

egorized as TAT-users and TAT-deficient, respectively,

based on the presence and absence of the essential com-

ponents of TAT machinery. The TAT users were again

classified into ‘‘minimal TAT user’’ and ‘‘extensive TAT

user’’ on the basis of whether the proportion of TAT-

dependent lipoproteins out of the total lipoproteins pre-

dicted was below or above 10%, respectively (see inset B

in Fig. 1). The bar diagram in Fig. 1 clearly shows that

among the TAT users, the majority of prokaryotes (398),

including those containing big genomes, are minimal users

of TAT. The rest (120) are extensive users, which also

includes prokaryotes with small genomes. As a specific

case, in eubacteria (with nearly equal numbers of small and

big genomes), the members with small genomes are

minimal TAT users and those with big genomes contain

both minimal and extensive users, irrespective of the size.

Hence there was no apparent correlation between the

number of TAT-dependent lipoproteins and the genome

size.

Though it is expected that the presence of the TAT

machinery would normally indicate the presence of TAT-

dependent lipoproteins, there were examples of TAT-con-

taining prokaryotes lacking TAT-dependent lipoproteins.

The majority of the archaeal genomes are small (46 out of

51) with either a few or no TAT-dependent lipoproteins.

Many (21 out of 51) lack detectable components of the

TAT machinery indicating that TAT is less significant for

lipoprotein targeting in archaea, which consists of organ-

isms living under extreme conditions. It was also found that

there was no apparent correlation between the number of

TAT substrates, which includes both lipoproteins and

nonlipoproteins (Dilks et al. 2003), and the number of

TAT-dependent lipoproteins. The largest prokaryote by

genome size, Sorangium cellulosum has 335 lipoproteins,

but only 24 (7%) of them were predicted to be exported via

the TAT pathway. In contrast, the relatively smaller pro-

karyote, Natronomonas pharaonis DSM 2160 was pre-

dicted to translocate 49 out of 67 (*73%) of its

lipoproteins via the TAT pathway.

Another parameter that defines prokaryotic genomes is

the base composition. As can be seen in the inset C in

Fig. 1, the extent of TAT usage for lipoproteins remains

uniformly low-to-moderate in organisms with 30–60% of

G ? C content. There is, however, a steep increase in TAT

usage among prokaryotes with very high G ? C content

(65–70%). Among Gram-positives, Firmicutes with low

G ? C content showed either no TAT components at all

(67% of 80 organisms) or only modest numbers of TAT-

dependent lipoproteins (32% of 80 organisms). Contras-

tingly 44 actinobacterial species (85% of 52 organisms)

with exceptionally high G ? C content (60–74%) were

predicted to be extensive TAT users.

Phylogenetic Analysis Suggests that TAT Utilization

for Lipoproteins Could Have Been Driven

by Adaptation to Complex Habitats

Generally, the TAT dependency appeared to be related to

the genera rather than the phyla. The archaeal phylum

Euryarchaeota consists of halophiles, methanogens, and

thermophiles. Among the 34 genera of Euryarchaeota for

which genome data was available, halophiles (five organ-

isms) showed 56–73% of their total lipoproteins to be TAT-

dependent; the highest was predicted for Natronomonas

pharaonis DSM 2160. Among methanogens, 17 out of 21

lacked TAT components; the rest of the methogens as well

as thermophiles (eight organisms) were predicted to utilize

J Mol Evol

123

TAT either poorly or moderately for lipoprotein biosyn-

thesis. Among the eubacteria, Firmicutes, the second largest

phylum consisting mainly of common pathogens like

Staphylococccus, Strepococccus, Clostridium, and Myco-

plasma, contain moderate-to-high numbers (30–100) of

lipoproteins, but uses TAT minimally between 0 and 3% of

total lipoproteins. However in this phylum, an exception to

this trend and a striking niche-based preference for TAT-

dependent lipoproteins was seen in the free-living bacteria,

Desulfitobacterium hafniense Y51 (21% of total lipopro-

teins) and Symbiobacterium thermophilum (17% of total

lipoproteins). In Actinobacteria, except two of the 54 gen-

era, the TAT-dependent lipoproteins, which are mostly

substrate-binding proteins, account for 15–25% of the total

lipoproteins. For example, Streptomyces ceolicolor, a soil-

dwelling bacterium and a prolific protein secretor, was

predicted to have significant numbers of TAT-dependent

lipoproteins (28% of total lipoproteins) including the proven

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MinimalTAT users

ExtensiveTAT users

Fig. 1 Eubacteria and archaea generally use TAT minimally: The

bar graph shows the distribution of 696 prokaryotes (bacteria and

archaea) arranged according to the phyla (horizontal axis), with

respect to the frequency (vertical axis on the left) genome size

categorization (vertical axis on the right), presence or absence of TAT

machinery and degree of usage (vertical axis on the right). The

categorization of genomes as small or big was decided on the basis of

the inflection point at 3.5 Mb seen in the genome size distribution

shown as inset A. The extensive and minimal users of TAT were

determined based on plotting the relative abundance of TAT-

dependent lipoproteins among total lipoproteins in an organism, as

shown in inset B. Those bacteria exceeding 10% were taken as

extensive users. Inset C shows the number of TAT-dependent

lipoproteins as a function of G ? C content of the genome

J Mol Evol

123

TAT substrates, peptidylprolyl cis–trans isomerase, a

putative sugar-binding protein, an iron–sulfur binding pro-

tein and a putative secretory protein (Widdick et al. 2006).

In Proteobacteria, the phylum with the maximum

number of genome sequences reported, 23,840 lipoproteins

have been predicted in 343 genomes but only 1,201 TAT-

dependent lipoproteins (ranging from 0 to 24% of the

lipoproteins, as the case may be) could be predicted. Chl-

amydiae and most of the Spirochetes even lacked detect-

able TAT components. We were, therefore, interested in

investigating the significance of TAT usage for lipopro-

teins across evolution and its influence in adaptation. From

the recent phylogenic tree obtained by comparing 31 uni-

versal protein families (Ciccarelli et al. 2006), we selected

the highly diversified Proteobacteria and matched the TAT

usage (%TAT-dependent lipoproteins among the total

lipoproteins). According to the phylogenetic analysis,

Delta, Epsilon, and a common ancestor for Alpha, Beta,

and Gamma subdivisions of Proteobacteria emerged from

Firmicutes, which are poor TAT users. However, even

among such phylogenetically related groups there was

significant difference in the usage of TAT for lipopro-

teins. For instance, the predominantly soil-dwelling Delta

proteobacteria are moderate users of TAT, whereas the

predominantly pathogenic Epsilon proteobacteria are poor

TAT users. In the Alpha, Beta, and Gamma proteobac-

terial group, Alpha and Beta proteobacteria contain free-

living and pathogenic bacteria in roughly equal numbers,

but Gamma proteobacteria are predominantly pathogenic.

Only the free-living organisms are moderate users of

TAT, whereas the pathogenic organisms use TAT mini-

mally (see Fig. 2). This trend indicates association of

TAT usage to an organism’s lifestyle rather than the

phylum it belongs to.

Functional Classification of Predicted TAT-Dependent

Lipoproteins

Many TAT substrates and the few lipoproteins identified so

far as TAT-dependent are either substrate-binding proteins

or cofactor-containing enzymes (Gimenaz et al. 2007;

Valente et al. 2007). To verify the possibility that TAT-

dependent lipoproteins could be a lifestyle adaptation of

prokaryotes in meeting the challenges posed by their sur-

roundings, the functional compatibility of such predicted

lipoproteins was investigated. Such functions should

obviously include digestive and detoxifying enzymes,

nutrient or metabolite transporting proteins, and compo-

nents of stress sensors and tolerance. One of the advantages

of lipid modification of a protein is its membrane locali-

zation providing access to the external milieu (Navarre and

Schneewind, 1999). As can be seen in the Pie-chart in

Fig. 3, 2307 TAT-dependent lipoproteins were predicted

from 518 genomes containing TatC and TatA/E. Among

these, 503 (22%) were enzymes, 562 (24%) were trans-

porters/binding proteins, and the remaining 54% were

either hypothetical proteins (647) or proteins with other or

unknown functions (595).

The distribution of these functional lipoproteins among

the minimal and extensive users according to genome size

is shown in Fig. 4. Enzymes, but not transporters/binding

proteins, are predominant among the minimal users,

whereas both are evenly distributed among the extensive

users. This interesting distribution has relevance to the

metabolic requirements according to their habitat. The

minimal use of the TAT pathway among the smaller gen-

omes is expected, as the host-obligates readily obtain their

nutrients from their respective hosts and therefore lipid

modification of the metabolizing enzymes would aid their

assimilation. On the other hand, extensive users of TAT

among small genomes are mostly extremophiles requiring

both transport/binding proteins and enzymes for nutrient

uptake and assimilation.

Predicted TAT-Dependent Lipoproteins as Transporters

and Binding Proteins

According to the analysis, components of ABC transporters,

RND efflux pump, TRAP transporters, and electron transfer

proteins were predicted as TAT-dependent lipoproteins in

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Without TAT-dependent Lipoproteins

With TAT-dependent Lipoproteins

Fig. 2 Prokaryotes surviving in complex habitats predominantly use

the TAT pathway for lipoprotein export: The bar graph shows the

distribution of bacterial genomes with predicted TAT-dependent

lipoproteins (filled bar) and without predicted TAT-dependent

lipoproteins (gray filled bar) surviving in complex habitats or host-

associated environments. Free-living bacteria surviving in complex

environments such as soil and aquatic habitats prefer TAT, with the

usage increasing with the complexity of the habitat. Pathogens and

other host-obligates use TAT lesser than free-living bacteria as they

depend on a host for survival. The data on lifestyle and habitat for the

microbial genomes was obtained from http://www.ncbi.nlm.nih.gov/

genomes/lproks.cgi

J Mol Evol

123

the extensive users of small and big genomes (Fig. 5). Out of

the 562 transporters/binding proteins, 194 are components

of ABC transporters, which constitute as high as 53% of the

total predicted TAT-dependent lipoproteins in small gen-

omes (predominantly from Haloarchaea) and 33% in big

genomes (mostly in soil bacteria of Actinobacteria). Among

the transporters/binding proteins of minimal users of big

genomes, the outer-membrane protein component of the

RND efflux pump (used in multidrug resistance mechanism)

is more common, especially in Gram-negative pathogens

like Burkholderia, Pseudomonas, Escherichia, Shigella, and

Xanthomonas. OprA of Burkholderia and OprM of Pseu-

domonas, which were predicted to be TAT-dependent

lipoproteins in this analysis, had been demonstrated to be

lipoproteins (Nakajima et al. 2000; Moore et al. 1999).

Substrate-/solute-binding proteins constitute 36% of the

total TAT-dependent transporter/binding lipoproteins. Such

lipoproteins include extracellular solute-binding proteins

Enzymes, 503, 22%

Superfamily unassigned hypothetical proteins, 228, 10%

Transporters and Binding proteins, 562, 24%

Superfamily assigned hypothetical proteins, 419, 18%

Other proteins, 595, 26%

Fig. 3 Transporters/binding proteins and enzymes predominate the

known functions of predicted TAT-dependent lipoproteins in pro-

karyotes: Pie-chart showing the functional distribution of 2,307

predicted TAT-dependent lipoproteins from bacterial and archaeal

genomes that contain a TAT machinery. The predicted TAT-

dependent lipoproteins are transporters/binding proteins, enzymes,

hypothetical proteins with superfamily-assignments and proteins of

unknown functions

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Oxido-reductases Hydrolases

Enzymes Transporters/Bindingproteins

0

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use

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Fig. 4 TAT-dependent

lipoprotein enzymes, rather than

transporters/binding proteins are

predominant among minimal

users (largely host-obligates),

the latter are preferred by

extensive users surviving in

complex habitats: The bargraph shows the functional

distribution of predicted TAT-

dependent lipoproteins

organized as functional class of

proteins (horizontal axis) in

relation to the frequency of

occurrence (vertical axis on the

left), the genome size and extent

of TAT usage (as defined in

Fig. 1, vertical axis on the right)

J Mol Evol

123

generally involved in chemoreception and transport of

nutrients and they fall in to either family-1 (specific to

oligosaccharides, a-glycerol phosphate, and iron) or family-

5 (specific to peptides and nickel, Tam and Saier 1993) of

binding proteins. Among the extensive TAT users of big

genomes, 72% of these proteins are contributed by soil

bacteria of Actinobacteria and 20% by sludge-dwellers of

Chloroflexi. Even among the smaller genomes, 60% of the

predicted TAT-dependent extracellular binding lipoproteins

belong to soil dwellers of Actinobacteria and 14% to

aquatic Cyanobacteria.

Predicted TAT-Dependent Lipoproteins as Enzymes

The next abundant functional class of predicted TAT-

dependent lipoproteins is enzymes. These are predomi-

nately hydrolases and oxido-reductases (typical TAT sub-

strates) (Fig. 6). Among small genomes 32 of the 180

predicted TAT-dependent lipoproteins were enzymes in

minimal TAT users, whereas only 43 of the 400 predicted

TAT-dependent lipoproteins were enzymes in extensive

TAT users. Among big genomes, there were 205 enzymes

among 800 TAT-dependent lipoproteins in the minimal

TAT user category and 223 enzymes out of 1000 TAT-

dependent lipoproteins predicted in the extensive TAT user

category. Hydrolases (128) predominate compared to

oxido-reductases (46) among the minimal users of big

genomes; both are almost equal in numbers among the

extensive users of big genomes (102 and 95, respectively)

as well as among the minimal users of small genomes (10

and 11, respectively).

Among the hydrolases, irrespective of genome size,

peptidases and esterases are the major types seen in mini-

mal and extensive users; lytic transglycosylases and

phosphatases are also major types in minimal users.

Hydrolases are commonly seen in pathogenic or symbiotic

bacteria. For instance, b-lactamases of Mycobacteria

and Burkholderia, that confer penicillin-resistance, were

predicted to be TAT-dependent lipoproteins; the one from

M. smegmatis has been experimentally verified to be TAT-

dependent (McDonough et al. 2005). Peptidases, phos-

phodiesterases, and alkaline phosphatases were the pre-

dominant types of hydrolases predicted as TAT-dependent

lipoproteins among marine bacteria and other free-living

bacteria. It is also interesting to note that bulk of the oxido-

reductases predicted as TAT-dependent lipoproteins among

the minimal users of big genomes belonged to one partic-

ular subclass, the DMSO reductases (26/177). The majority

of these were molybdenum containing DMSO reductases

of Shewenella sps.

Hypothetical Proteins belong to Mostly Substrate-

Binding Proteins and Enzymes such as Hydrolases

and Oxido-Reductases

Out of 647 hypothetical proteins, 419 could be assigned to

SCOP domains using the superfamily database (Wilson

et al. 2009). The significant entries indicated that like the

known TAT-dependent lipoproteins, the predicted func-

tional domains also belonged to the categories of enzymes,

substrate-binding proteins and transport proteins (Fig. 7).

Among enzymes, a fairly even distribution of hydrolases

(38%) and oxido-reductases (35%) were seen, followed by

transferases (14%), isomerases and lyases (4%). Periplas-

mic-binding protein-like I and II superfamilies (44%)

predominated the functional class of binding proteins and

transporters. Cytochromes and electron transport proteins

(14%), components of the RND efflux pump (7%) and

metal-binding proteins, predominantly copper- and iron-

binding proteins were also predicted. It is intriguing that

domains of outer-membrane lipoprotein localization fac-

tors were exclusively assigned to hypothetical TAT-

dependent lipoproteins of Gram-positive Bacillus species.

Even the hypothetical TAT-dependent lipoproteins turned

out to be oxidoreductases, hydrolases and binding proteins

by SCOP domain assignment.

ABC-Transporters, 194, 35%

RND-Efflux, 58, 10%TRAP-Transporters, 24, 4%

Electron transfer proteins, 49, 9%

Iron-enterobactins, 9, 2%

Substrate/Solute- Binding proteins, 206, 36%

Other Binding and transport proteins, 22, 4%

Fig. 5 Substrate/solute-binding proteins and components of ABC

transporters form majority of transporters and binding proteins among

the predicted TAT-dependent lipoproteins: The pie-chart shows

functional distribution of predicted TAT-dependent lipoproteins as

transporters/binding proteins. In addition to ABC transporters, the

outer-membrane component of the RND efflux system, electron

transport proteins, iron-enterobactins and components of TRAP

transporters are also predicted as TAT-dependent lipoproteins

J Mol Evol

123

Oxido Reductases, 177, 35%

Hydrolases, 255, 50%

Transferases, 18, 4%

Lyases, 9, 2%Ligases, 35, 7%

Isomerases, 9, 2%

Peroxidases/SOD, 13, 7%DMSO Reductases,

26, 15%Fumarate Reductases,

14, 8%Thioredoxins, 10, 6%

Molybdopterins, 24, 14%

Other Oxidoreductases, 90, 50%

Peptidases/Proteases, 57, 23%

Esterases/Deacetylases/Lipases, 53, 21%

Beta-lactamases, 28, 11%Phosphatases/Diphosphatases/Phosphohydrolases, 34, 13%

Lytic transglycosylases, 39, 15%

Other hydrolases, 44, 17%

A

B

Fig. 6 Majority of the

predicted TAT-dependent

lipoproteins as enzymes are

hydrolases and oxido-

reductases: Pie-chart shows

distribution of enzyme classes

among predicted TAT-

lipoproteins. Other enzyme

class, transferases, isomerases,

ligases, and lyases are also

predicted as TAT-dependent

lipoproteins. The majority is

further grouped into sub-

classes; inset A shows

subclasses of oxido-reductases

and inset B shows subclasses of

hydrolases as predicted TAT-

lipoproteins

Enzymes, 201, 49%

Transporters and binding proteins, 98, 23%

Hypothetical proteins, 10, 2%

Proteins with other functions, 110, 26%

Periplasmic binding protein, 42, 44%Cytochromes and Electron

transport proteins, 14, 14%

Other binding proteins, 13, 13%

RND efflux, 7, 7%

Lipoprotein localization factors, 5, 5%

Cupredoxins, 5, 5%

ABC transporter, 5, 5%

Ferritin - like and HIPIP 4, 4%

Other metal - binding proteins, 3, 3%

Hydrolases, 76, 38%

Oxidoreductases, 69, 35%

Transferases, 28, 14%

Isomerases, 8, 4%Lyases, 7, 4%

Ligases, 9, 5%A

B

Fig. 7 Hypothetical TAT-

dependent lipoproteins with

superfamily-assignments are

enzymes, substrate-binding

proteins, and transport proteins:

Hydrolases and oxido-

reductases contribute to the

majority of enzymes class.

Periplasmic-binding protein-

like I and II superfamilies

predominate the functional class

of binding proteins and

transporters. Cytochromes and

electron transport proteins,

RND efflux pump component

and copper- and iron-binding

proteins are also predicted

among this functional class

J Mol Evol

123

Discussion

Knowledge about bacterial lipid modification has been

gradually advancing with the identification of several

bacterial lipoproteins and from the recent studies of lipo-

protein biosynthesis and targeting (Sankaran and Wu 1994;

Selvan and Sankaran 2008; Hutchings et al. 2009). In fact,

this understanding has led to the design of strategies for

converting nonlipoproteins to lipoproteins that could have

potential biotechnological applications such as ELISA,

biosensors, vaccines, targeted-drug delivery and also in

display-libraries (Kamalakkannan et al. 2004). The

recently identified translocation system (TAT pathway),

except for indirect experimental evidence (Valente et al.

2007), remains inadequately explored for its role in the

export of bacterial prolipoproteins. As a first step, the

prevalence and significance of TAT-dependent lipoproteins

in 696 prokaryotic genomes were investigated using the

new prediction tool ‘‘TATLIPO.’’ This was developed

based on the currently accepted prediction tools, TatP and

DOLOP for TAT substrates and bacterial lipoproteins,

respectively. In the absence of an experimentally verified

dataset, its predictability, as judged by the correlation of

predicted TAT-dependent lipoproteins to the presence of

TAT machinery in prokaryotes was 95% [(696 - 34)/696].

However, the prediction of TAT-dependent lipoproteins in

34 prokaryotes lacking TAT machinery and no predicted

TAT-dependent lipoproteins in 127 prokaryotes possessing

TAT machinery indicated an overall accuracy of prediction

of 77%. It is noteworthy that in cases where TAT com-

ponents, TatC and TatA/E are missing, on the average only

one or two TAT-dependent lipoproteins were identified, of

which 60% were hypothetical. In cases where the TAT-

dependent lipoproteins were not predicted even though

TAT components were present, the TAT usage was found

to be limited, averaging only five TAT substrates per

organism compared to the overall average of 27 per

organism, as deduced using TatP for 696 prokaryotic

genomes.

According to Bolhuis (2002), the proteins of halophiles

tend to fold rapidly and are thereby rendered Sec-incom-

patible; the higher TAT usage of halophiles among the

extremophiles of Euryarchaeota could be an attempt to

escape protein aggregation and precipitation at high intra-

cellular salt concentrations (4–5 M of K?). Similarly such

selection pressures could have driven lipoproteins to

become TAT-dependent. In another instance, Firmicutes

contain mostly pathogens and minimal users of TAT.

However, there are two exceptions that signify the role of

TAT-dependent lipid modification in environmental adap-

tation. Desulfitobacterium hafniense Y51, a dehalorespiring

firmicute thrives in the complex milieu, tetrachloroethene-

contaminated soil, by virtue of the presence of a large

number of specialized electron donors and acceptors

(Nonaka et al. 2006), many of which are TAT-dependent

lipoproteins. The Firmicute, Symbiobacterium thermophi-

lum, a marine thermophile with a high G ? C content, is an

extensive TAT user (Sugihara et al. 2008).

For lipoproteins in general a direct correlation between

the number and the genome size, had been reported (Babu

et al. 2006). However, no correlation could be observed

between the number of TAT-dependent lipoproteins and

the genome size, the number of lipoproteins or the number

of TAT substrates. This is similar to the case of proteins

involved in the secretion and transport of inorganic ions

and such an adaptation in response to environmental sig-

nals was found not to correlate with the genome size

(Konstantinidis and Tiedje 2004). However, the increase in

TAT usage for lipoproteins in prokaryotes with high

G ? C content is interesting and points to habitat-based

adaptation, as these are normally free-living in complex

habitats like soil. According to Bentley and Parkhill

(2004), free-living prokaryotes possess larger genomes and

are equipped to survive in a complex and variable envi-

ronment whereas, pathogenic bacteria in general have

smaller genomes, as they can readily acquire nutrition from

their stable niche. This correlation is reflected in our phy-

logenetic analysis of the largest phylum, Proteobacteria, in

which, irrespective of the phylogeny, pathogenic bacteria

appears as poor users of TAT, while the free-living and soil

bacteria are moderate-to-extensive users. To scavenge

nutrients from such competitive and variable habitats,

prokaryotes depend largely on high-affinity substrate-

binding proteins of transporters, as seen in the ABC

transport machinery (Albers et al. 2004). For instance,

Actinobacteria, which comprises essentially of soil-dwell-

ing bacteria, has been predicted to be an extensive TAT

user for lipoprotein biosynthesis, especially for substrate-

binding lipoproteins. Such high-affinity binding at the

surface of the organism is essential for an efficient response

to the amount and quality of available nutrients in a

complex and altering milieu and to even physico-chemical

conditions (Roszak and Colwell 1987).

Electron transfer is an essential bioenergy metabolism

intimately coupled to nutrient utilization. Therefore, their

coupling should be efficient to maximize the chances of

surviving in challenging conditions. Halocyanin, a copper-

containing lipoprotein is involved in the transfer of elec-

trons in high-salt habitats. The halocyanin isolated from

Natronomonas pharaonis contains characteristic N-acetyl

S-diphytanyl Cys as the N-terminal amino acid similar to

that of eubacteria (Mattar et al. 1994). Interestingly,

our analysis has also predicted that these halocyanins are

TAT-dependent lipoproteins, suggesting the role of TAT-

dependent lipoproteins for survival in high-ionic environ-

ments. Similarly, in Shewanella, significant numbers of

J Mol Evol

123

ferredoxins that mediate transfer of electrons in several

metabolic reactions are predicted to be TAT-dependent

lipoproteins. Being anaerobic inhabitants of sediments or

deep sea environments, this genus employs ferredoxins for

respiration while utilizing a variety of terminal electron

acceptors such as nitrate, nitrite, thiosulfate, sulfite etc.,

abundant in such habitats (Heidelberg et al. 2002).

The lifestyle adaptation of prokaryotes is exemplified by

the presence of lipid-modified digestive enzymes tethered

to the surface for effective accumulation of nutrients (Na-

varre and Schneewind 1999). The presence of hydrolases

among extensive users of TAT signifies the digestive and

nutrition acquisition capabilities. Peptidases, phosphodies-

terases, and alkaline phosphatases, predicted in marine and

free-living bacteria are essential to metabolize organic

matter in ever-changing mineral rich habitats. For instance,

the inorganic phosphate content in marine habitats is low

(3 nM) and therefore alkaline phosphatases are required to

liberate phosphate from organic phosphates (Hassan and

Pratt 1977). Oxidoreductases are the other major class of

enzymes predicted as TAT-dependent lipoproteins and are

involved in detoxification and downstream metabolism of

nutrients. The DMSO reductases of Shewanella sp. are

essential for extracellular respiration of DMSO, which is

abundant in aquatic environments due to its release by

algae and other microplanktons. The enzyme has been

found localized to the cell surface of Shewenella to enable

efficient capture of DMSO from the complex habitat

(Gralnick et al. 2006). It is noteworthy that our analysis had

predicted this proven lipoprotein as TAT-dependent and

thus pointing to niche-based adaptation. The requirement

of such lipoproteins, especially at the cell surface to

metabolize substrates, reveals the significance of TAT-

dependent lipid modification in environmental adaptation.

In a similar lifestyle adaptation (Cases et al. 2003), the

larger number of transcriptional regulators identified in

free-living bacteria rather than in intracellular pathogens

and endosymbionts, was attributed to rapid adaptation to

environmental conditions.

Taken together, the predicted functional profile of TAT-

dependent lipoproteins closely matches the expected

functional attributes necessary for the adaptation of a

prokaryote to its surroundings, particularly providing an

advantage to survive under challenging conditions.

Implications of the Present Finding in Lipoprotein

Biosynthesis and Applications

Bacterial lipoprotein biosynthesis has been exploited by

our group and a few others to engineer proteins and express

them as lipoproteins for a variety of applications such as

enhanced antigenicity, outer surface display, and liposomal

integration. For instance, the use of bacterial lipid

modification as a novel protein engineering method was

successfully demonstrated by Kamalakkannan et al. (2004)

and it has opened-up promising applications. However,

these strategies if based on the erroneous premise that all

lipoprotein biosynthesis and translocation depend on the

Sec-machinery, would be incompatible with prefolded

proteins. Therefore, the new protein engineering design for

such fast-folding or prefolded proteins would have to

involve fusion of TAT-dependent lipid modification signals

in the signal peptide. Similarly the major aims of metabolic

engineering include providing or enhancing the viability of

an organism in harsh environments or to utilize specific

compounds for useful metabolic conversions. Such appli-

cations would most probably require fast folding substrate-

binding proteins or cofactor-requiring enzymes on the

surface of a bacterium. TAT-dependent lipoprotein modi-

fication could provide a convenient strategy to engineer

such target proteins for such applications, as in the adap-

tation that appears to be working in nature. Already,

uptake-hydrogenases, hydrogenase-1 and hydrogenase-2

(known TAT substrates) have been targeted for enhanced

production of hydrogen in E. coli (Maeda et al. 2007). The

database created from the current analysis can be useful to

select such targets.

Conclusion

Prompted by the presence of TAT-signal sequence in

bacterial lipoproteins, an extensive and up-to-date com-

putational analysis of possible role of TAT in lipoprotein

export across the membrane was undertaken. It revealed

that many of these are substrate-binding proteins, trans-

porters, enzymes that could be a useful adaptation for

viability and survival in a niche. These predicted TAT-

dependent lipoproteins are mostly present in free-living

prokaryotes in complex environments or niche, a striking

example being Haloarchea; host-obligates hardly use TAT

for lipoprotein transport. Though there is no correlation

with the genome size or phylogeny a rough correlation is

seen with respect to G ? C content. There is a tendency to

use TAT extensively with increasing G ? C content. Our

findings also have implications for potential protein engi-

neering applications. This comprehensive study reveals the

significance of the TAT pathway in lipoprotein biosyn-

thesis and its understanding is essential in developing

versatile protein and metabolic engineering tools.

Acknowledgments We thank A. Tamil Selvan for the algorithm

and script. We are grateful to Prof. Edward J. Behrman, Ohio State

University, USA for his critical inputs to improve the readability of

the manuscript and Prof. Venkat Gopalan, Ohio State University,

USA for his valuable help. UGC-DRS and DBT-Center of Excellence

programmes are acknowledged for financial support and fellowship to

J Mol Evol

123

HS. MMB acknowledges the Medical Research Council, UK, Darwin

College Cambridge and Schlumberger Ltd. for support.

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