It has long been appreciated that bacteria communicate with each other using chemical signal molecules. These molecules are critical in coordinating gene expression and synchronizing the activities of the entire community. The term “quorum sensing” (QS) refers to regulation of gene expression in response to fluctuations in cell-population density. QS bacteria constitutively produce, release, detect, and respond to chemical signaling molecules called autoinducers which generally accumulate as the cells grow in number (Fuqua et al., 2001; Miller and Bassler, 2001; Schauder and Bassler, 2001). Depending upon the bacterial species, various physiological processes are mediated by these cell–cell communication systems, including antibiotic production, virulence, symbiosis, conjugation, and biofilm formation (Schauder and Bassler, 2001).

In the past decades, one of the most well-studied QS signaling molecules is N-acyl homoserine lactone (AHL) which is mainly found in Gram-negative bacteria (Williams et al., 2007). AHL molecules are highly conserved among bacteria. Each AHL molecule consists of a homoserine lactone ring unsubstituted in the β- and γ-positions but N-acylated at the α-position with a fatty acyl group. The structure of AHL shows variation in acyl chain length (varies from C4 to C18), the degree of saturation, and the oxidation states (presence of a hydroxy-, oxo-, or no substituent at the C3 position; Chhabra et al., 2005). Hence, it is the fatty acyl group that confers QS signal specificity among the bacteria (Cooley et al., 2008). AHL molecules are synthesized by LuxI homologue synthase. When AHL concentration has reached its threshold level, the AHLs will bind to their cognate receptor (LuxR homologue), thereby stimulating the expression of numerous downstream target gene. Each LuxR-type protein is highly specific for its respective AHL. There are multiple QS circuits that control a myriad of specific genes that express many bacterial phenotypes and potential virulence determinants (Parsek and Greenberg, 2000).

Dong et al. (2002) reported that the biochemical mechanism of action of the LuxI/LuxR pairs is conserved in many bacterial species. In most cases, further regulatory complexity has been added to the basic biochemical mechanism. This allows a multitude of behaviors to be functioned and controlled by a common mechanism. This can be seen in opportunistic human pathogen Pseudomonas aeruginosa in which two LuxI/R pairs were found (called LasI/LasR and RhlI/RhlR) that work simultaneously to control the expression of genes involved in biofilm formation and virulence (Passador et al., 1993; Brint and Ohman, 1995; Glessner et al., 1999). Besides its role in many physiological processes, the bacterial QS is also essential in developing pathogenic relationships with eukaryotic hosts. Hence, the AHL signaling system has been regarded as a promising target for developing novel approaches to interfere with microbial QS by regulating the virulence of the entire population. This indirectly creates a less selection pressure for the evolution of antibiotic-resistance in the bacteria (Hentzer and Givskov, 2003).

The genus Acinetobacter comprises aerobic, Gram-negative, non-fermentative bacteria that are isolated from diverse environments (Bhargava et al., 2010). As of now, at least 14 complete genomes of Acinetobacter species are available in the database, and only three environmental isolates have been sequenced (Kim and Park, 2013). A flurry of research over the past decade has focused mainly on A. baumannii strains because of their clinical importance as the primary pathogenic bacteria in nosocomial infections. Hence, the role of QS system in soil-borne or plant-associated Acinetobacter sp., which have been only rarely explored, are a valuable line of study.

Our group have been exploring rhizosphere environment for bacterial communities in the Malaysian rainforest. Recently, we isolated Acinetobacter sp. strain GG2 from rhizosphere of ginger (Zingiber officinale; Chan et al., 2009, 2011) and its genome was fully sequenced by Illumina platform (Hong et al., 2012) and deposited in GenBank. This soil isolate was found to secrete only long chain AHLs, particularly 12-carbon in acyl chain length with different variant. In the present study, we aimed to analyze the genome assembly for gene predictions and annotations, as well as comparative genome analysis with other closest sequenced Acinetobacter spp. The annotated genome led us to the investigation of the putative homologue of AHL synthase, designated as aciI. The aciI gene was cloned and overexpressed in Escherichia coli and the purified protein was characterized. Mass spectrometry confirmed the production of AHLs was directed by the recombinant AciI protein.

With the availability of the whole-genome and AciI of isolate GG2, these genome data will path the way for functional study of QS in GG2 in the future. As such, the verification of the synthase activity provides a platform to study the regulatory role of the AHLs on virulence and unknown genetic traits of the soil-dwelling bacterium. In addition, Acinetobacter species isolated from diverse environments may have profound biotechnological potential such as degradation of a variety of pollutants as they appear to be metabolically versatile (Jung et al., 2010). As the frequency of multidrug-resistance among Acinetobacter strains is increasing (Bhargava et al., 2010), QS as the global regulator is gaining more importance as the target for antimicrobial strategies to attenuate bacterial virulence. It is reported that within the genus, some of the Acinetobacter environmental species are genetically closely related to the clinical sources (Gonzalez et al., 2009). Hence, this facilitates the study of QS mechanism in environmental strains of Acinetobacter sp. which could also be applied to the pathogenic isolates.

In previous work by Hong et al. (2012), the Illumina HiSeq 2000 platform (Illumina Inc., San Diego, CA, USA) was employed to perform the whole-genome sequencing of strain GG2. The genome sequence has been deposited at DDBJ/EMBL/GenBank under the accession no. ALOW00000000. With approximately 56 fold coverage, the genome assembly generated 57 contigs with a total of 3,890,805 bp. The genome has G+C content of 38.4%. From Prodigal analysis, a total of 3,572 coding DNA sequences (CDS) were predicted and the genome coding density is 88%.

Annotations by RAST revealed a total of 419 subsystems with 48% subsystem coverage. A subsystem represents a collection of functional roles that make up a metabolic pathway, a multi-subunit complex (e.g., the ribosome) or a specific class of proteins (e.g., signal transduction). Meanwhile, subsystem coverage shows the percentage of the FIGfams (a set of proteins that are “globally similar” and in which all members share a common function) that is covered by subsystems (Aziz et al., 2008). Among the features of the subsystems, at least two third encode the basic core functions and metabolic pathways of the organism. The most abundant of the subsystems are related to amino acids and derivatives (n = 441, 16.8 of total subsystem features), followed by carbohydrates (n = 272, 10.4%), cofactors, vitamins, prosthetic groups, pigments (n = 226, 8.6%), protein metabolism (n = 209, 8.0%), RNA metabolism (162, 6.2%), and fatty acids, lipids and isoprenoids (154, 5.9%). From the analysis, other closely related A. baumannii species were also found to have high abundance of CDS coding for carbohydrate and amino acids and derivatives (Table S1). In addition, RAST annotation shows that A. baumannii AB0057 (score 537), A. baumannii AYE (score 532), A. baumannii ACICU (score 530), and A. baumannii ATCC 19606 (score 516) are the closest neighbors of the strain GG2.

Interestingly, we identified several unique subsystem features of strain GG2 from RAST analysis (Figure 1). Similar to Acinetobacter ACICU and AYE strains, strain GG2 might utilize D-ribose and fructose as the major carbon source, besides having serine-glyoxylate cycle for one-carbon compound metabolism. This environmental bacterium also does not rely on siderophore in iron acquisition system, unlike other closely related Acinetobacter sp. Reflecting to its non-environmental lifestyle of strain GG2, the bacterium was found to encode less CDS for antibiotic-resistance compounds in comparison to pathogenic A. baumannii AB0057 and AYE strains. This is evident from the annotated genome that strain GG2 does not encode for aminoglycoside resistance genes as found in other strains. We also found several environmental important genes encoding extracellular lipase and cell wall-degrading enzymes such as endoglucanase. In metabolism of aromatic compounds, strain GG2 possesses genes for quinate degradation, a feature which was solely found in this organism. Similar to other Acinetobacter sp. except A. baumannii 19606, strain GG2 does not encode gentisate 1,2-dioxygenase responsible for degradation of xylenols and cresols. In contrast, genes involved in photosynthesis or motility and chemotaxis in strain GG2 and other A. baumannii strains were not identified in this study.

Once gene annotation was performed, we were interested to look at the genome similarity between strain GG2 and other Acinetobacter species. Using BRIG software, the analysis of the genome sequence of strain GG2 showed high homology with at least 70% identity of the genome similarity with four closely related A. baumannii strains (Figure S1). Meanwhile, genomic alignments using MAUVE program indicated that strain GG2 shows a high degree of genome synteny with completely sequenced multi-drug resistant strain, A. baumannii AB0057 (Figure 2). The colored blocks of A. baumannii AB0057 are connected by lines to the homologous regions in the colored blocks of strain GG2. There are not many ‘white space’ which denote sequences not in homology blocks, hence showing the two species share a large number of their genome sequences. Areas that are completely white within a colored block are not aligned and they may contain sequence elements specific to a particular genome. The study on the genome assembly was then narrowed down to mainly CDS responsible for cell-to-cell communication among proteobacteria. Analysis of the LuxI gene clusters shows a conserved variation among strain GG2 with other close relatives of Acinetobacter sp. (Figure 3). All the Acinetobacter strains studied possess luxI homologues with presence of upstream transcriptional regulator, luxR homologues in reversed orientation. In the vicinity of the LuxI/R genes are long chain fatty acid coA ligase, acyl-CoA dehydrogenase, major facilitator superfamily transporter and enoyl CoA hydratase, all which are required in fatty acid synthesis.

Web-based similarity searches against the GenBank database indicated that AciI protein sequence is highly homologous to other AHL synthase and several proteins from other Acinetobacter species with 96% sequence identity with AHL synthase from A. calcoaceticus. In addition, AciI protein shares 88% identical residues with several members of LuxI family from Acinetobacter sps. In fact, the multiple sequence alignments revealed that AciI protein shares high homology as well as the 10 conserved amino acids with other reported autoinducer proteins of Acinetobacter sps. as shown in Figure 4. On the other hand, the phylogenetic tree constructed based on amino acid alignment (Figure 5) illustrated that AciI was clustered closely with a hypothetical protein from Acinetobacter sp. NIPH 973 with bootstrap value of 87%. However, AciI was found to be the least phylogenetically related to AHL synthase from A. oleivorans DR1 and a hypothetical protein from Acinetobacter sp. NIPH817, possibly that these Acinetobacter species get diverted in evolution.

The putative AHL synthase, aciI, was found in contig 3 of the draft genome and its sequence has been deposited in the GenBank database (Accession number ALOW01000034.1). From NCBI database and Figure S2, this ORF encodes for a protein which consists of 183 amino acids. The sequence, TAAAG, at 32 nucleotides upstream from the start codon and the sequence, TTACCG, located at 60 nucleotides upstream correspond to the potential –10 and –35 transcription sequences, respectively. There are 17 nucleotides separating the two consensus regions, in agreement to the optimum spacing suggested by Hawley and McClure (1983) on E. coli promoter analysis. A putative Shine-Dalgarno site (AAGC) is located 8 bp upstream from the start codon while the transcription initiation site is postulated to be 7 bp downstream of –10 region. At the downstream part of the gene, there is a sequence likely to be a rho-independent transcription termination sequence from nucleotide 883 to nucleotide 1013 which consists of an inverted repeated sequence with the potential to form a hairpin structure with a 10-nucleotide loop and a 10-nucleotide stem including four pairs of CGs. Interestingly, a putative lux-box (CTGTAAATTTTTACAG) for strain GG2 was found 74 bp upstream of the start codon or immediately upstream of –35 element. This sequence is highly similar to the lux-box of A. baumannii M2 and was postulated to be the binding site for LuxR homologue protein, designated AbaR (Niu et al., 2008).

The 552 bp aciI was amplified by PCR (Figure 6A) and cloned into pET28a overexpression vector, producing pET28a-aciI, with a 6× His-tag driven by a T7 promoter. E. coli BL21 was transformed with this recombinant plasmid and the recombinant aciI gene was overexpresed upon IPTG induction. The His-tagged recombinant protein was later purified using affinity chromatography using nickel-chelated sepharose column (Figure 6B). By following the reading frame and prediction from ExPASy server (Wilkins et al., 1999), the theoretical isoelectric point (pI) of the recombinant protein is 5.37 and a molecular weight of 20.5 kDa. Nevertheless, the AciI protein expressed in E. coli cells was a fusion protein with 6× His-tag peptide, resulting in a protein with an estimated total molecular weight of 24 kDa. This is in accordance with the SDS PAGE profile of the purified recombinant protein as demonstrated in Figure 6B. Most of the recombinant protein was found in the cytoplasmic fraction of the cell lysate.

The extracted AHL from the spent culture supernatant of the IPTG-induced E. coli BL21 harboring pET28a-aciI was analyzed using Agilent 6490 Triple-Quad LC-MS/MS system. High-resolution mass spectrometry analysis demonstrated the presence of long chain AHLs, 3-oxo-dodecanoyl-homoserine lactone (3-oxo-C12-HSL) and 3-hydroxy-dodecanoyl-homoserine lactone (3-hydroxy-C12-HSL) with m/z values of 298.1000 and 300.1000, respectively (Figure 7). The mass spectra of the extracted AHL were indistinguishable to the corresponding synthetic compounds at their respective retention times. Both AHLs were not found in the E. coli BL21 harboring pET28a alone or pET28a-aciI in non-induced state. The mass spectra also revealed quantitatively that 3-hydroxy-C12-HSL was produced more abundantly than 3-oxo-C12-HSL after 8 h of induction.

Acinetobacter spp. are ubiquitous in nature, and commonly present in the soil, water, sewage and sediment environments, indicating the profound adaptability of this genus in different niches. They are also known as effective degraders of alkanes and aromatic hydrocarbons (Throne-Holst et al., 2007; Fischer et al., 2008). In drinking water, these Gram-negative bacteria tend to form aggregates (Simoes et al., 2008). However, some Acinetobacter species including A. baumannii are pathogenic and multi-drug resistant strains. In fact, A. baumannii is historically regarded as an opportunistic pathogen in which its ability to cause diseases is determined by major deficiencies in the immunocompromised patients rather than the intrinsic virulence determinants of the infecting strains (Joly-Guillou, 2005).

In this study, we deciphered the draft genome of GG2 and comparative genomic analysis was performed with its closest sequenced relatives. According to RAST analysis, an important point worth noting is the high similarities between strain GG2 and A. baumannii AB0057, A. baumannii ACICU, A. baumannii AYE, and A. baumannii ATCC 19606. These four closest species are primarily associated with large outbreaks of nosocomial infections isolated from hospitalized patients (Manchanda et al., 2010). The 3.89 Mb genome of strain GG2 has G+C content of 38.4%, a value close to 40% which corresponds to that reported for other members of the Acinetobacter genus (Vallenet et al., 2008). The genome size of strain GG2 was slightly smaller than its closely related species as it encodes for less CDS responsible for basic survival needs of the soil isolate such as carbohydrate metabolism and some virulence factors. These virulence factors were found primarily in some multi-drug resistant Acinetobacter species but not in strain GG2. A study has been shown that human clinical isolate A. baumannii AYE was found to harbor 86-kb genomic island, a drug resistance island present in the majority of published A. baumannii genomes (Adams et al., 2010). Up to date, some of the non-pathogenic soil-living Acinetobacter sps. which have been completely sequenced are A. oleivorans DR1, A. baylyi ADP1, and A. calcoaceticus PHEA-2 (Jung et al., 2011). Similar to these environmental strains, GG2 was found to have a broad range of metabolic capacities as demonstrated by RAST analysis. With such feature, this rhizospheric bacterium is well-adapted in nutrient acquisition in soil and rhizosphere ecosystems. We also observed relatively high number of genes involved in expressing cofactors, vitamins, prosthetic groups and pigments, suggesting the ability of this bacterium to cope with various growth conditions and stresses in diverse ecological niches.

As for carbohydrate metabolism, besides ability to utilize fructose and D-ribose, it is postulated that strain GG2 is able to metabolize glucose using Entner–Doudoroff pathway (EDP). CDS encoding key enzymes for EDP such as gluconokinase (gntK) and phosphogluconate dehydratase (edd) were found from the draft genome. Other key enzymes such as 2-keto-3-deoxy-6-phosphogluconate aldolase (eda) and enzymes involve in the phosphorylation of gluconate (i.e., gntP) were not found, possibly the CDS fall in the sequence gaps of the genome. The presence of a number of CDS which encodes enzymes responsible for exopolysaccharides synthesis is another additional feature of a soil-living organism.

In addition, RAST analysis confirms the absence of high affinity iron-binding molecules called siderophore. Without such phenotype, it is postulated that strain GG2 depends on hemin transport system to scavenge iron from its environment. This may reflect a competitive advantage of the bacterium to obtain iron to thrive in different kinds of rhizosphere environment. A study by Yamamoto et al. (1994) reported that when Acinetobacter sp. invades the host, one of the mechanism of persistence and toxicity is the iron acquisition system, a likely contributing factor in its pathogenesis. This possibly explains the ability of strain GG2 to establish its niche in rhizosphere environment. On the other hand, a substantial number of genes were found to be associated with degradation of aromatic compounds, particularly on degradation of quinate, an aromatic plant compound. This is supported by the presence of quinate dehydrogenase which was found in the annotated genome. This physiological attribute enables the bacteria to degrade the metabolites synthesized by the host plants. Apart from this, analysis by RAST revealed the annotated genes were 48% of the subsystem coverage. Further annotation and bioinformatics analysis on hypothetical proteins could shed light on the functional roles of proteins with unknown functions and may reveal novel proteins that confer a fitness advantage to strain GG2 within the host rhizosphere. These proteins could be crucial in plant-microbe interaction as the role of strain GG2 as endophyte or phytopathogen in ginger rhizosphere remains unknown.

The genomes of strain GG2 was aligned with A. baumannii AB0057, A. baumannii ACICU, A. baumannii AYE, and A. baumannii ATCC 19606 using BRIG software (Figure S1). The high similarity among the Acinetobacter sps. indicates a close relationship among the bacterial strains. Hence, it is believed that the four A. baumannii nosocomial strains may possibly be evolved from environmental strains such as strain GG2 or they may share some ancestry relationship. Analysis by MAUVE (Figure 2) indeed showed a high degree of synteny between strains GG2 and its closest species, A. baumannii AB0057, in agreement with BRIG analysis. In silico analysis of the luxI gene cluster among strain GG2 and its closely related species showed conserved LuxI/R QS-related genes (Figure 3) among the environmental and nosocomial pathogenic strains. A point worth noting is the presence of fatty acid synthesis-related genes which are found at both upstream and downstream regions of LuxI/R homologues. Such profound feature was also reported by Kang and Park (2010) in LuxI/R gene cluster of Acinetobacter sp. strain DR1. This may indicate the use of metabolites of fatty acid biosynthetic machinery as the precursors for autoinducer proteins to form AHL lactone ring and the acyl group in strain GG2 and its close relatives (Val and Cronan, 1998). However, such mechanism remains unknown and requires further validation.

In this work, the gene for putative AHL synthase from Acinetobacter GG2, designated as aciI, has been successfully cloned and characterized in this study. The recombinant protein was fused with His-tag peptide to facilitate the purification of the protein. The purified protein was in agreement with the estimated size from SDS-PAGE analysis. The deduced protein sequence was highly similar and conserved to several AHL synthases from other Acinetobacter spp. Analysis of the draft genome sequences revealed that AciI is highly to be the only member of the LuxI family in Acinetobacter sp. GG2 genome as there is no additional gene that encodes LuxI homologue. The multiple sequence alignment and phylogenetic tree constructed (Figures 4 and 5) illustrated a high degree of homology and conserved regions among AHL synthases from other Acinetobacter spp. All the strains shared the 10 invariant amino acids which are characteristics of LuxI homologues (Parsek et al., 1997). This strongly indicates a low rate of random mutation for this autoinducer gene. It also shows that these proteobacteria share similar basic QS mechanism and gene regulation in AHL synthesis although they are responsible for different target genes.

A detailed analysis of both upstream and downstream sequences of aciI gene found that although both –10 and –35 promoter regions are not strongly conserved, the sequences meet the requirement of the typical E. coli RNA polymerase σ⁷⁰ consensus promoter sequences (Hawley and McClure, 1983; Harley and Reynolds, 1987). In addition, the prokaryotic transcription termination sequences were present downstream of the stop codon. The presence of such features at the promoter and downstream regions of the ORF serve as strong indications that the aciI mRNA is likely a monocistronic transcript, and therefore is transcribed independently of other genes and artificial factors.

When E. coli harboring the recombinant aciI was induced with IPTG for 8 h and its spent supernatants was assayed with LC-MS/MS, the presence of both long chain AHLs, 3-oxo-C12-HSL and 3-hydroxy-C12-HSL was confirmed, suggesting the AciI is indeed the AHL synthase of Acinetobacter sp. GG2 (Figure 7). Such findings are in consistent with a recent study by Chan et al. (2011) which obtained the same AHL profile in Acinetobacter sp. GG2. The production of 3-hydroxy-C12-HSL was much higher than 3-oxo-C12-HSL, possibly indicating the important role of the former AHL in executing the physiological functions of the cells or expressing virulence factors.

In the past decade, the role of autoinducer proteins in Acinetobacter sp. was widely explored. One of the earliest studies on AHL synthase produced by Acinetobacter was conducted by Niu et al. (2008). The gene, designated as abaI (EU334497), was found in A. baumannii strain M2, a major human nosocomial infectious pathogen. The study demonstrated the importance of AbaI in normal biofilm formation for the bacteria to survive under unfavorable growth conditions. An abaI null mutant was shown to be impaired in biofilm forming capabilities by 40% after 16 h of growth in comparison to its wild type strain, and this was restored when AHL was supplied externally, indicating that there is a direct role of AHL molecules in biofilm development (Niu et al., 2008). Nevertheless, the mechanism of QS in contributing to the virulence and pathogenic potential in these bacteria is yet to be known.

The association of AHL and biofilm formation was first demonstrated by McLean et al. (1997). The study showed the production of bacterial AHL in aquatic biofilms growing on submerged stones, but was not present in rocks lacking a biofilm (McLean et al., 1997). In contrast to other QS systems, the AHL-mediated QS signaling system in numerous bacterial species appears to control genes associated with colonization of eukaryotes and this process was shown to be facilitated by bacterial biofilms (Costerton et al., 1999). A recent study by Anbazhagan et al. (2012) found that ∼60% of the clinical isolates of Acinetobacter spp. showed a significant biofilm formation with production of AHL molecules under prolonged period of incubation. In fact, Niu et al. (2008) reported a knockout mutant of abaI homologue was shown to have inhibit formation of biofilm. In another study, QS-regulated gene expression was shown to play a vital role in the metal tolerance of biofilms in A. junii strain BB1A. In the presence of natural or synthetic QS inhibitor, the growth of strain BB1A leading to the biofilm formation in metal-supplemented medium was significantly inhibited with a longer lag phase (Sarkar and Chakraborty, 2008).

Interestingly, many Acinetobacter spp. show some varying AHL profile. A study by Gonzalez et al. (2001) on A. calcoaceticus BD 413 (an environmental strain) and two clinical isolates from hospitalized patients demonstrated that multiple signaling molecules with autoinducer activity were detected in each Acinetobacter strain. In another study, a set of 43 Acinetobacter strains from nosocomial and environmental sources were studied and it was shown that 63% of the bacterial strains produced more than one AHL. However, none of the AHL signals could be specifically assigned to a particular species of the genus Acinetobacter (Gonzalez et al., 2009).

In a recent report, Bitrian et al. (2012) performed analysis of virulence markers on nine hospital and environmental strains of Acinetobacter sp. and found that all the strains studied secreted medium to long-chain AHLs. No short chain (C4–C6) AHLs were detected in any case. This is a distinctive feature of strains belonging to the A. calcoaceticus – A. baumannii complex. The pathogenic A. baumannii strain M2 has been shown to produce a major AHL molecule 3-hydroxy-C12-HSL, directed by autoinducer synthase abaI. Although five additional minor AHLs (e.g., unsubstituted C10-HSL, C12-HSL, 3-hydroxy-C10-HSL, unsaturated 3-oxo-C11-HSL, C14-HSL) were detected in culture supernatants of this strain, only one AHL synthase gene was identified, suggesting that this synthase has low specificity and is capable of synthesizing other QS signals (Niu et al., 2008). In another study, a LuxI homologue, termed as AqsI, was identified from diesel-degrading Acinetobacter sp. strain DR1 (Kang and Park, 2010). Similar to AciI, this AqsI protein consists of 183 amino acids and was found to secrete C12-related AHLs. This is consistent with the findings that C12-related AHL was a major QS signal in A. baumannii strain M2 (Niu et al., 2008). The study by Kang and Park (2010) also demonstrated that ability of aqsI mutant of strain DR1 in producing biofilm and degrading hexane were reduced remarkably. However, the restoration of the mutant phenotype was observed after the addition of free wild-type cell supernatant and exogenous C₁₂-AHL, indicating the importance of QS in bacterial communication (Kang and Park, 2010).

The most studied Acinetobacter spp. are clinical isolates which are mostly isolated from hospitalized patients. Hence, the genetics and molecular biology available from the environmental strains of Acinetobacter spp. is not well-documented. Nevertheless, phylogenetic tree analysis (Figure 5) in fact showed that the environmental isolate (i.e. strain GG2) are clustered with many clinical isolates. The high similarities among the clinical and environmental strains highly suggest that the environment is an ideal habitat for opportunistic human pathogens, especially the nutrient-rich rhizosphere, the zone around roots that is influenced by the plants. According to Berg et al. (2005), the features that make a bacterial strain an efficient plant growth promoter (e.g., antagonistic properties, colonization ability) could also make it an etiological agent in bacterial opportunistic infections.

In this present study, the genome of strain GG2 is a significant addition of the genomic data from the Acinetobacter genus and offers different prospects into how closely related organisms are successful in different environments. As information of bacterial traits determining its ability in host plants colonization is still limited, detailed analysis of the genome sequence of strain GG2 will help to shed some light in prediction of the roles of the bacterium in the rhizosphere environment. In addition, the cloning and characterization of aciI as the homologue of AHL synthase of Acinetobacter sp. GG2 represents the initial step in elucidating the precise role and the molecular mechanism of the autoinducer system possessed by the bacterium. Among other things, this work now makes it possible to construct mutants with defective aciI to determine the roles of AHL in Acinetobacter sp. GG2, and to study the interaction of this LuxI homologue with molecules demonstrating anti-QS properties. As such, this work provides an impetus for further investigation of the relationship of AHL, QS and quorum quenching in this rhizospheric isolate. On a final note, data from further studies also helps to shed some light on the role of strain GG2 and its adaptation in its niche of rhizosphere environment.

KYH and KGC conceived and designed the experiments; KYH performed the experiments and analyzed the data; KYH and KWH wrote the paper; CLK, CKS, WFY and KGC edited and approved the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.