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Brazilian Journal of Microbiology

Print version ISSN 1517-8382

Braz. J. Microbiol. vol.42 no.1 São Paulo Jan./Mar. 2011 



Comparative analysis of two component signal transduction systems of the Lactobacillus acidophilus group



Yanhua CuiI,*; Xiaojun QuII

ISchool of Food Science and Engineering, Harbin Institute of Technology Harbin 150090 P. R. China
IIInstitute of Applied Microbiology, Heilongjiang science academy, Harbin, 150010, P. R. China




The Lactobacillus acidophilus group is a phylogenetically distinct group of closely related lactobacilli. Members of this group are considered to have probiotic properties and occupy different environmental niches. Bacteria generally sense and respond to environmental changes through two component systems (TCSs) which consist of a histidine protein kinase (HPK) and its cognate response regulator (RR). With the use of in silico techniques, the five completely sequenced L. acidophilus group genomes were scanned in order to predict TCSs. Five to nine putative TCSs encoding genes were detected in individual genomes of the L. acidophilus group. The L. acidophilus group HPKs and RRs were classified into subfamilies using the Grebe and Stock classification method. Putative TCSs were analyzed with respect to conserved domains to predict biological functions. Putative biological functions were predicted for the L. acidophilus group HPKs and RRs by comparing them with those of other microorganisms. Some of TCSs were putatively involved in a wide variety of functions which are related with probiotic ability, including tolerance to acid and bile, production of antimicrobial peptides, resistibility to the glycopeptide antibiotic vancomycin, and oxidative condition.

Key words: Lactobacillus acidophilus group, two component system, histidine protein kinase, response regulator protein, bioinformatics analysis




The Lactobacillus acidophilus group ("acidophilus complex") is a phylogenetically distinct group of closely related lactobacilli, containing, among others, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus delbrueckii subsp. bulgaricus (30). Several members of this group are considered to have probiotic properties. Strains of L. acidophilus, L. johnsonii, and L. delbrueckii subsp. bulgaricus have been extensively studied for their probiotic activities, pathogen inhibition, epithelial cell attachment, and immunomodulation. Members of these species can inhibit pathogen, prevent intestinal tract infections, improve the immune system, and reduce inflammatory or allergic reactions (5, 20, 29). At the same time, L. acidophilus and L. delbrueckii subsp. bulgaricus have the ability to alleviate lactose intolerance. So they have not only been widely used in the manufacture of fermented dairy but are also consumed as probiotic products (5, 20, 29).

Survival during passage through the gastrointestinal (GI) tract of humans is generally considered a key feature for probiotics to preserve their expected health-promoting effects (5, 11). Survival of microorganisms during their transit through the GI tract requires the ability to sense and respond to the various and changing conditions present in the environment. Bacteria generally sense and respond to environmental changes through two component systems (TCSs). TCSs are present in the majority of Gram-positive and Gram-negative bacteria, and are one of the most important mechanisms for external environmental sensing and signal transduction (15, 37). TCSs are involved in controlling a wide variety of physiological processes, such as chemotaxis, biofilm formation, stress, osmolarity, quorum sensing, and virulence (15, 38). A typical TCS consists of a membrane-associated histidine protein kinase (HPK) and a cytoplasmic response regulator (RR). The former detects specific environmental signals and the latter regulates expression of genes.

Although the L. acidophilus group has received much attention in the past few years and some L. acidophilus group genomes have recently been sequenced and published (1, 19, 27, 35), only little research has been done on TCSs in this bacterial group. Only recently, LBA1524/LBA1525 in L. acidophilus was found to be related with acid tolerance, and LBA1430/LBA1431 with bile tolerance (3, 26). Since so little is known about TCSs in the L. acidophilus group, we scanned TCSs in five genomes of this group and predicted function of putative TCSs. At the same time, we compared the differences between five members of this group based on TCSs.



Sequence information

Complete genome sequences of L. acidophilus NCFM, L. gasseri ATCC 33323, L. johnsonii NCC533, L. delbrueckii subsp. bulgaricus ATCC 11842, L. delbrueckii subsp. bulgaricus ATCC BAA365, and Lactobacillus plantarum WCFS1were obtained from the National Center for Biotechnology Information (NCBI,

Sequence analysis

Protein domain organizations were determined by SMART and Pfam (4, 31). TMHMM 2.0 was used to detect transmembrane helices (17). Multiple sequence alignments were created using the CLUSTAL X software (34). The evolutionary distances were calculated using the software package TREECON. The phylogenetic tree was generated by the neighbour-joining method using the software package TREECON (36).

Identification of HPKs and RRs

The genome sequences of the L. acidophilus group were searched for genes encoding putative HPKs and RRs by means of HMMER2.3.2 ( Consensus Protein Families Database ( sequences for HisKA (PFAM00512), HATPase_c (Pfam02518), and Response_reg (PFAM00072) were used in HMMER searches of each genome. The HisKA HMM and HATPase_c HMM were used to scan for the phosphoryl-accepting domain and highly conserved HATPase domain of HPKs, while the Response_reg HMM was used to scan for the highly conserved phosphoryl accepting domain of RRs. Putative functions were assigned to target genes manually by sequence comparison to an existing protein database using the NCBI protein-protein BLAST server ( blast/blastp).



The two component systems in the L. acidophilus group

The five completely sequenced genomes of the L. acidophilus group, including L. acidophilus NCFM, L. gasseri ATCC 33323, L. johnsonii NCC533, L. delbrueckii subsp. bulgaricus ATCC 11842 and ATCC BAA365 were scanned in order to predict TCSs by means of the Pfam HMMs HisKA, HATPase_c and Response_reg. The L. plantarum WCFS1 genome was predicted in the same way for comparative analysis. L. plantarum is a flexible and versatile species that is encountered in a variety of environmental niches, including some dairy, meat, and many vegetable or plant fermentations. This flexible and adaptive behavior is reflected by the relatively large number of regulatory and transport functions, including 13 TCSs (16).

The five to nine putative HPKs containing a conserved histidine residue and a C-terminal HATPase domain, and five to nine putative RRs containing a RR receiver domain were detected in the genomes of the L. acidophilus group (Table 1). 14 HPKs and 15 RRs were predicted in L. plantarum WCFS1, which is significantly higher than those of the L. acidophilus group. The difference of the genomes' size is one of reasons. L. plantarum and the L. acidophilus group species, respectively, belong to facultative heterofermentatives, and obligately homofermentatives. L. plantarum has a comprehensive sugar metabolism. Its genome encodes all enzymes required for the glycolysis and phosphoketolase pathways. L. plantarum displays heterolactic fermentation or homolactic fermentation, depending on the environmental conditions. However, the members of L. acidophilus group have only glycolysis pathway and display homolactic fermentation. It is proposed that differences in their metabolism are linked to the number of TCSs. L. plantarum need more regulation and signaling systems in order to perform the different metabolism. At the same time, the results likely reflect adaptations of the L. acidophilus group to the stable and nutritionally rich milk environment or human gut, where fewer biosynthetic functions and less adaptive regulation are required.

Most of the TCSs are arranged in pairs, either with the HPK-encoding gene first followed by the RR-encoding gene, or vice versa. L. delbrueckii subsp. bulgaricus ATCC11842 contained a HPK and a RR gene not encoded in pairs. However, there is not single HPK or RR in the genome of ATCC BAA365. This difference was likely related to the origin of strains. The strain ATCC11842 was originally isolated from bulgarian yogurt by S. Orla Jensen in 1919, while the strain ATCC BAA365 was derived from a French starter culture CHCC757. Strains of different origin have adapted differently to fit specific niches during evolution. Similar results were obtained in thermophilic lactic acid bacteria Streptococcus thermophilus (28). Based on comparative genome hybridization analysis of 47 dairy S. thermophilus strains, the researchers revealed variable gene composition among S. thermophilus strains. It was presumed that there were frequent recombination or gene transfer within S. thermophilus, and some genes have disappeared and degenerated in the stable environment. At the same time, we find that the numbers of TCSs in S. thermophilus LMD-9 are smaller than those in strains CNRZ1066 and LMG18311.

Classification of HPKs and RRs

For all HPKs and RRs detected, the protein domain organization was analyzed using TMHMM, Pfam and SMART. The results of these analyses are shown in Fig 1 A and 1B. All HPKs are classified in 7 groups according to protein domain organizations. Most of the HPKs are predicted to be membrane localized, consistent with the observation that localization of the sensor kinase to the membrane of the bacterial cells appears to be a general feature of most TCSs (38). Many of these HPKs contain previously described domains, including PAS domain, PAC domain and HAMP domains. PAS (Per-ARNT-Sim) domains monitor changes in redox potential, cellular oxygen, overall energy level of a cell, light, and small ligands. This domain is found in proteins regulating circadian rhythms and hypoxia responses as well as in input domains for TCSs. PAS domains are frequently followed by a 40-to 45-amino-acid PAC motif (33). PAS/PAC domains are widely distributed but are found primarily in proteins involved in signaling or regulation of transcription. The groups 6 and 7 contained PAS domain, including LGAS_0065, LJ0066, LBA0079, Ldb0136, Ldb0963, LBUL_0112 and LBUL_0873. HAMP domain is found in several ATP-binding proteins, for example histidine kinase, DNA gyrase B, topoisomerases, heat shock protein HSP90, phytochrome-like ATPases and DNA mismatch repair proteins (2). HAMP domains are found in the groups 3, 5 and 6. The precise functions of HAMP domains are unknown, however, mutation in the HAMP repeat region of Neurospora crassa is responsible for the most severe osmosensitivity and dicarboximide resistance phenotypes (21).

All RRs are classified in 3 groups according to protein domain organizations. Most output domains of RRs belonged to Trans_reg_C domain. The LJ1659 from L. johnsonii contains a typical LuxR-type HTH motif at the C terminus of proteins. This domain is a DNA-binding, helix-turn-helix (HTH) domain of about 65 amino acids, present in transcription regulators of the LuxR/FixJ family of response regulators. LuxR-type HTH domain proteins occur in a variety of organisms. LuxR-type HTH regulators control a wide variety of activities in various biological processes, such as bioluminescence, virulence, spore formation, acetate metabolism (8-9, 13).

The LJ0766, LJ0918 of L. johnsonii, the LBA1798, LBA0603 of L. acidophilus and the LBUL_0021, Ldb0026 of L. delbrueckii subsp. bulgaricus contain LytTR type output domain. The LytTR domain is a DNA-binding, potential winged helix-turn-helix domain (~100 residues) present in a variety of bacterial transcriptional regulators of the algR/agrA/lytR family (23). LytTR domain is a type of DNAbinding domain and different from helix-turn-helix or wingedhelix type output domain. This domain is distributed widely in low G+C Gram positive bacteria, and is involved in biosynthesis of extracellular polysaccharides, quorum sensing, bacteriocin peptide production (7, 18).

The putative HPKs and RRs were grouped in order to predict the subfamily of HPKs and RRs. Two bootstrapped NJ trees were constructed, an HPKs tree and an RRs tree (not shown). The HPKs tree was constructed with the L. acidophilus group HPKs phosphotransferase domains and highly conserved HATPase domain, while the RRs tree was constructed with all RR receiver domains.

In addition to this initial set of sequences, homologous sequences of other bacterial species were included to improve the resolution of both trees. Based on the two trees, the L. acidophilus group HPKs and RRs were classified into the subfamilies described by Grebe et al. (15). The results of classification are shown in Table 2. The HPKs typically contain two functionally and structurally distinct parts, a variable N-terminal sensor region and a conserved C-terminal kinase core domain. The latter have highly conserved residues called homology boxes, including the H-, N-, D-, F-, and Gboxes. The conserved boxes are presumed to play crucial roles in substrate binding, catalysis, and/or structure. Based on the presence and structure of the various homology boxes, Grebe and Stock made a comprehensive classification of HPKs (15). According to the criteria, the putative HPKs fell into five subfamilies (1a, 2a, 3a, 7, and 10). Most HPKs are members of the subfamily HPK1a. This is the most common type HPK. The LJ1658 was unique and was found to belong to subfamily HPK7. The HPK7 subfamily has the following characteristics: the H-box is distinguished by the presence of a negatively charged group 2 residues upstream from the conserved histidine and a positively charged residue, usually an arginine, 8 residues upstream. The F-box is missing and the distance between the D-and the G-boxes is reduced. The LBA0602, LBA1799, LJ0448, LJ0764 and LBUL_0022 were classified into subfamily HPK10. The HPK10 subfamily members commonly possess five to seven N-terminal transmembrane segments, and have no D-box. The subfamily 10 HPKs usually are related with quorum sensing.

Based on Grebe classification scheme, the putative RRs were grouped into 3 subfamilies (14-15). Most RRs were found to belong to OmpR subfamily. This subfamily appears to be the most abundant subfamily of RRs in the Gram positive bacteria whose genomes have been sequenced to date. The LBA0603, LBA1798, LJ0449, LJ0766, LBUL_0021, and Ldb0026 belonged to LytR subfamily, while the LJ1659 from L. johnsonii belonged to FixJ subfamily. Analysis of the two trees showed that the receiver domains of all RRs pairing to a HPK of a certain subfamily generally clustered together in the same branches of the RRs tree. For example, all RRs pairing with a subfamily HPK10 contained a HTH-DNA-binding domain of the LytTR family. These results were consistent with previous research that the HPK phosphotransferase domains, the cognate receiver domains and the RR output domains have evolved as integral units (15).

Function prediction of HPKs and RRs

To get functional annotation of the HPKs and RRs, bootstrapped NJ trees of HPKs and RRs were constructed with whole sequences of HPKs and RRs respectively (Fig 2-3). The phylogenetic analysis revealed nine major groups of the L. acidophilus group HPKs (Fig. 2). Five HPK groups (I, III, VII, VIII, IX) contain closely related sequences from all L. acidophilus group examined (Fig. 2). Thus, the sequences within each group are conserved in the L. acidophilus group studied, may represent orthologs with common functions and likely involved in basic adaptation for environment. In contrast, the groups II, IV, V, VI contain the sequences from only some members. The results implied that the group II, IV, V, and VI were special for some members of the L. acidophilus group.

Lactobacillales-specific clusters of orthologous protein coding genes (LaCOGs) had been built using computational procedures in 12 sequenced Lactobacillales genomes (19). Most HPKs in group V belonged to LaCOG01758, including lp_0416, lp_3063, lp_1355, lp_3581, lp_3088, LJ0448, and LJ0764. The lp_0416 and lp_3581of L. plantarum were involved in QS, so we predicted the group V was related with QS. This result was consistent with previous research (10, 32). The HPKs in group IX belonged to LaCOG00289. The Nterminal domains of HPKs in group IX contained a HAMP domain and a PAS/PAC domain. The domain structure of group IX was similar to that of Enterococcus faecalis VicK (25). So these HPKs were likely related to resistance to glycopeptide antibiotics vancomycin.

The phylogenetic RRs tree revealed 9 major groups for the L. acidophilus group RRs (Fig. 3). Analysis of the two trees showed that all RRs pairing to a HPK of a certain group generally clustered together in the same branches of the RRs tree.

To get a more specific functional annotation of the L. acidophilus group HPKs and RRs, they were compared with those of other bacterial species, using the NCBI BLAST server. Maintaining an E-value cut-off of 1e-44, we found a number of the L. acidophilus group TCSs to be similar to systems with a known biological function (Table 2, Suppl. Fig.1).

Survival of bacteria is an important first step in the colonization of and probiotic contribution to the GI tract (5). The gastric acidity is the main obstacle to survival of bacteria. So the capacity of a microorganism to tolerate acidic pH is essential to the production and functionality of a probiotic culture. The TCS LBA1524/LBA1525 from L. acidophilus is identified to respond to acid (3). The insertional inactivation of the LBA1524 gene was found to reduce cell survival in pH 3.5. Thus the LBA1524/LBA1525 may aid the persistence and survival of microbes in an acidic environment. The TCSs LGAS_1397/LGAS_1398 and LJ1630/LJ1631 showed high identity to LBA1524/LBA1525. The HPKs and RRs of these TCSs showed 55%-56% and 85% identity to LBA1524 and LBA1525, respectively. L. delbrueckii subsp. bulgaricus is widely used as starter culture in the manufacture of yogurt and fermented milk products, so the strain need to tolerate the low pH in milk fermentation. The Ldb1492/Ldb1493 and LBUL_1388/LBUL_1389 in L. delbrueckii subsp. bulgaricus show high similarities to the LBA1524/LBA1525. The above mentioned TCSs were predicted to tolerate high acidic environment.

Because these Lactobacillus strains reside in the intestines, they must tolerate the presence of bile to survive in this environment. Bile is a multifaceted stressor, which can disrupt cell membranes and cause damage to DNA and proteins. The LBA1430/LBA1431 from L. acidophilus has been previously identified to respond to bile stress (26). We could also identify some TCSs putatively involved in tolerance bile. These TCSs are similar to LBA1430/LBA1431, including LGAS_0711/LGAS_0710, LJ1586/LJ1587, Ldb0878/Ldb0877 and LBUL_0803/LBUL_0802. The presence of these TCSs in the L. acidophilus group indicates an adaptation to the GI tract, enabling the bacteria to survive the acidic and bile-rich environments of the stomach and small intestine.

One of the properties of a probiotic strain is the ability to produce antimicrobial substances such as bacteriocins. L. acidophilus and L. johnsonii can produce a number of different bacteriocins, including lactacin F, lactacin, Acidophilucin A, and lactacin F. The genomes of L. acidophilus and L. johnsonii have revealed operons coding for bacteriocins. The bacteriocins may have an important role in inhibiting pathogenic bacteria of the human gut. We could also identify TCSs from L. johnsonii and L. acidophilus putatively involved in bacteriocin production and resistance. The LBA0602/LBA0603, LBA1799/LBA1798, LJ0448/LJ0449, and LJ0764/LJ0766 are similar to AbpK/AbpR which plays important role in the production of class II bacteriocins ABP118 in L. salivarius (12). The LJ0448/ LJ0449 is unique and different from other putative TCSs relating to bacteriocin production. The GC content of the LJ0448 and LJ0449 genes are 22.4% and 24.3%. These GC contents are significantly lower than the average value of 34.6% observed for the entire L. johnsonii NCC533 genome. Interestingly, the LJ0448/ LJ0449 genes have not been found in other bacterial genomes, using Blastb. The results indicate that it may have been acquired recently via horizontal gene transfer.

We could also identify some TCSs from each member of the L. acidophilus group examined putatively involved in susceptibility to the glycopeptide antibiotic vancomycin (Table 2). These TCSs showed similarities to VicK/VicR in Enterococcus faecalis and HPK48/RRP48 in L. sakei (22, 25). Vancomycin is widely used to treat severe infections by Grampositive bacteria. In lactic acid bacteria, the mechanism of resistance to vancomycin remains to be elucidated, although some lactic acid bacteria showed resistance to vancomycin.

Among putative TCSs, the TCS LJ1658/LJ1659 from L. johnsonii is unique. LJ1658 belonged to subfamily HPK7, and LJ1659 contained a FixJ type output domain. Although a vast majority of QS-TCSs comprise HPK10 type HPK and LytR type RR, ComP/ComA of Bacillus subtilis is a QS-TCS that belong to HPK7 and FixJ type RR (36). So we presumed LJ1658/LJ1659 was possibly involved in QS. The TCS was similar to a system of unknown function (SMU.1965c/ SMU.1964c) of Streptococcus mutans (6). Furthermore, the genes encoding the TCSs LJ1658/LJ1659 and SMU.1965c/ SMU.1964c appeared to share strong gene neighbourhood conservation. Based on the neighbouring genes, which encode putative (sugar) periplasmic transport systems, these TCSs were putatively involved in host-microbe interactions, functioning in the utilization of nutrients in host.

By scanning five members of the L. acidophilus group genomes for TCSs, we have gained information about the capacity of these probiotic microorganisms to adapt to changes in their environment. The five to nine TCSs were predicted in the five genomes. These TCSs were involved in adapting to specific environment (e.g., acid tolerance, bile tolerance, aerobic/anaerobic respiration), resistance to glycopeptide antibiotics vancomycin, and production of bacteriocin. The results presented here provide a basis for future research on signal transduction mechanisms in the L. acidophilus group. At the same time, the results showed some TCSs were conserved in the L. acidophilus group, and other TCSs were specific for some lactic acid bacteria. The distribution of TCSs in the L. acidophilus group showed these lactic acid bacteria had adapted differently to fit their specific niches.



This work was supported by Natural Scientific Research Innovation Foundation in Harbin Institute of Technology (HIT. NSRIF. 2008.19), National Nature Science Foundation of China (Grant No.30901048), and Development Program for Outstanding Young Teachers in Harbin Institute of Technology (HITQNJS. 2007. 36).



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Submitted: April 22, 2009; Approved: August 23, 2010.



* Corresponding Author. Mailing address: School of Food Science and Engineering, Harbin Institute of Technology Harbin 150090 P. R. China.; E-mail:

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