Introduction

The genus Microvirga was established by Kanso and Patel (2003), which describes aerobic, Gram-negative, non-sporulating and rod-shaped bacterium, which required yeast extract for growth. The first species described in the genus Microvirga was Microvirga subterranea (Kanso and Patel 2003); the genus was assigned to the class Alphaproteobacteria, phylum Proteobacteria. At the time of writing this manuscript, the genus Microvirga comprised nine species (LPSN, http://www.bacterio.net/microvirga.html). Members of Microvirga have been isolated from various habitats, e.g., Japanese hot spring (Takeda et al. 2004), Australian geothermal waters (Kanso and Patel 2003), Chinese rice field soil (Zhang et al. 2009), Korean atmospheric samples (Weon et al. 2010), cow pea grown in semiarid Brazil (Radl et al. 2014), and nitrogen fixing Lupinus texensis from Texas, USA (Ardley et al. 2012). Recently, another one as-yet not validly named new species, Microvirga massiliensis sp. nov., was isolated in Marseille from a stool sample collected in Senegal, and it had the human commensal with the largest genome (Caputo et al. 2016).

During investigation of the microbial diversity of desert soil of Cholistan, Bahawalpur, Pakistan, several strains including pink colored strain designated NCCP-1258T were isolated. Based on 16S rRNA gene sequence analysis, strain NCCP-1258T was most closely related to Microvirga lotononidis (type strain WSM3557T) (Ardley et al. 2012), which was isolated from native legumes Listia angolensis (in Zambia) and Lupinus texensis (Texas, USA). In this study, a bacterium, designated NCCP-1258T, was characterized by polyphasic taxonomic approach to delineate its exact taxonomic position. Further, phenotypic and biochemical characterization was performed along with phylogenetic relationships of 16S rRNA gene and four housekeeping genes.

Materials and methods

Isolation, morphology and phenotypic characterization

During a study of bacterial diversity from desert soil of Cholistan, Bahawalpur, Pakistan, (lat/lon 29°23′43″N 71°41′1″E) a soil sample was serially diluted in sterilized water, and inocula from 10−3 to 10−4 dilutions were spread on R2A agar medium (containing yeast extract 0.1 %, peptone 0.1 %, casein hydrolysate 0.1 %, soluble starch 0.1 %, glucose 0.1 %, sodium pyruvate 0.06 %, KH2PO4 0.06 % and bacto agar 1.5 %) and incubated at 40 °C. During isolation, a pink-colored colony of strain NCCP-1258T was recovered after 3 days of incubation on R2A agar medium at 40 °C. For further purification, the strain was streaked repeatedly. The purified cells of strain NCCP-1258T were maintained on R2A medium and stored in glycerol stocks at –80 °C as well as in lyophilized ampules. The type strains of closely related species, Microvirga lotononidis WSM3557T (=LMG 26455T), M. lupini Lut6T (=LMG 26460T), M. zambiensis WSM3693T (=LMG 26454T) and M. flocculans ATCC BAA-817T (=JCM 11936T) were used as reference strains in all the characterization experiments unless otherwise mentioned.

Growth of strain NCCP-1258T was tested on various media, such as ISP 2, oat meal agar (ISP 3), TSA, R2A and nutrient agar media at 40 °C. The colony morphology of strain NCCP-1258T was observed on R2A agar at 40 °C after 3 days of incubation. Cells grown on R2A agar for 24 h were observed using phase-contrast microscopy (BH-2; Olympus) and further detailed morphology under scanning electron microscopy (QUANTA 200; FEI). Gram staining was carried out using the standard Gram reaction (Gregersen 1978). Growth at various temperatures (4, 10, 15, 20, 28, 30, 33, 37, 40, 45, 50, 55 and 60 °C) was observed on R2A agar for 1 week. The pH range for growth was tested at pH between 4.0 and 11.0 (with 0.5 pH value increments) using the buffer system described by Xu et al. (2005) at 40 °C for 4 days in R2A broth and the growth was determined using a spectrophotometer at OD600 nm. Tolerance to NaCl (0–20 %, w/v, with 1 % increment) was investigated on R2A agar by incubation at 40 °C for 10 days. Catalase and oxidase activities were determined as described previously (Kovacs 1956). Growth under anaerobic conditions was determined on R2A agar supplemented with or without 0.1 % nitrate by using the GasPak Anaerobic Systems (BBL) according to the manufacturer’s instructions.

The biochemical and enzymatic activities and utilization of sole carbon and nitrogen sources were determined using API 20E, API 50CH, API 20NE, and API ZYM strips according to the manufacturer’s instructions (bioMérieux, France). Further physiological and biochemical features of strain NCCP-1258T were determined using Biolog GN III microplate™ by incubating at 40 °C according to the manufacturer’s instructions.

Phylogenetic analyses

PCR amplification and sequencing of 16S rRNA gene of strain NCCP-1258T were performed using the protocol described previously (Li et al. 2007). The phylogenetic position of strain NCCP-1258T was identified based on 16S rRNA gene sequence and by comparison with the sequences of type species on EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/ (Kim et al. 2012) and BLAST search on the DDBJ/NCBI servers. To clarify the taxonomic status of the strain, housekeeping genes [gyrase subunit B (gyrB), RNA polymerase beta subunit (rpoB), deoxyribonucleic acid subunit K (dnaK) and recombination protein subunit A (recA)] were also amplified and sequenced using the primers and annealing temperature conditions as described by Ardley et al. (2012). The sequences were submitted to DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/) and are listed in respective dendrograms.

Phylogenetic analyses were performed using MEGA 6 software package (Tamura et al. 2013) based on 16S rRNA gene sequences of strain NCCP-1258T and its closely related taxa. Phylogenetic trees were constructed using three algorithms: maximum parsimony (MP), neighbor joining (NJ), and maximum likelihood (ML) methods. The phylogenetic relationship was also confirmed using housekeeping loci. The sequence similarities of the housekeeping genes were estimated with the available sequences of closely related validly published species using the Kimura 2-parameter model. The concatenated data set was created by combining the nucleotide sequences of the four housekeeping genes, and phylogenetic trees were reconstructed using this concatenated data set with the concatenated sequences of related species of the genus Microvirga and other closely related genera. The stability of the relationship was assessed using bootstrap analysis for all the phylogenetic trees with 1000 re-samplings for the tree topology.

DNA base composition, DNA–DNA hybridization

DNA–DNA hybridization was performed between strain NCCP-1258T and the reference strains. Total genomic DNA was extracted using a combination of the protocols of Marmur (1963) as described previously (Goris et al. 1998). DNA–DNA hybridizations were performed with biotin-labelled probes in microwell plate (NUNC) according to the method of Ezaki et al. (1989) with modifications by Goris et al. (1998) and fluorescence measurements were conducted using Bio Assay Reader (model HTS7000, PerkinElmer). The hybridization was performed at 40 °C with eight replications. The DNA G+C content of the genomic DNA of strain NCCP-1258T was determined on reversed-phase HPLC of enzymatically degraded DNA of Escherichia coli DH5α as the reference (Mesbah et al. 1989).

Chemotaxonomy

Chemotaxonomic characteristics of strains NCCP-1258T and its reference strains were determined under the same experimental conditions. Cellular fatty acid analysis was performed by growing strain NCCP-1258T and all reference strains on TSA at 33 °C for 3 according to the recommendation given by Sasser (1990) using Microbial Identification System (Sherlock version 6.1; MIDI database: TSBA6). Menaquinones were extracted and analyzed using HPLC by following the procedures of Collins et al. (1977) and Kroppenstedt (1982). Polar lipids were extracted and identified by two-dimensional thin-layer chromatography by following procedures of Minnikin et al. (1979) and Collins and Jones (1980).

Results and discussion

Morphology and phenotypic characterization

The 3-day-old cells of strain NCCP-1258T were pleomorphic with round ends, which occurred singly, in pairs or in small chains or clusters (Supplementary Figure 1a–e). Optimum growth of cells was observed in R2A medium with pH 7.5–8.5 (range 6.5–9.5), at 40 °C (range 20–45 °C) and could tolerate 0–2 % NaCl (optimum 0.5 %, w/v). Cells of strain NCCP-1258T were non-motile, which differentiated strain NCCP-1258T from the closely related reference species: M. flocculans, M. zambiensis and M. lotononidis that are reported to be motile by polar flagella (Takeda et al. 2004; Ardley et al. 2012). Strain NCCP-1258T was positive for oxidase and urea hydrolysis, unlike M. flocculans and M. subterranean which did not hydrolyze urea, M. zambiensis, M. lupini, M. lotononidis, and M. subterranean were oxidase negative. NCCP-1258T was positive for tryptophan deamination, whereas the closely related strains were either weakly positive or negative (Kanso and Patel 2003; Takeda et al. 2004; Ardley et al. 2012). Strain NCCP-1258T hydrolyzed esculin and gelatine (weak) and produce acid from arbutin and d-melibiose, but the closely related strains did not (Ardley et al. 2012). Other detailed reactions which differentiated strain NCCP-1258T from the closely related species of Microvirga are enlisted in Table 1 and/or described in species description.

Table 1 Differentiating phenotypic and biochemical characteristics of strain NCCP-1258T with the type strains of closely related species of genus Microvirga
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Phylogenetic analysis, DNA base composition and DNA–DNA hybridization

The comparison of 16S rRNA gene sequence (1499 nucleotides; DDBJ/EMBL/GenBank accession number LC065285) of strain NCCP-1258T showed the highest similarity of 98.0 % with Microvirga lotononidis WSM3557T, 97.4 % with M. vignae BR3299T, 97.2 % with M. lupini Lut6T and M. zambiensis WSM3693T and 97.1 % with M. flocculans ATCC BAA-817T and less than 97 % with the other species of genus Microvirga and other taxa of the related genera on the EzTaxon Server database. A neighbor joining phylogenetic trees based on 16S rRNA gene and concatenated sequences of four housekeeping genes (Fig. 1, Supplementary Figure 2) revealed that strain NCCP-1258T fell within the radiation of a cluster comprised of Microvirga lotononidis, M. vignae, M. lupini, M. zambiensis and M. flocculans with a bootstrap value of 72 %. A similar tree topology was also observed when the phylogenetic analyses were performed using MP and ML algorithms (Fig. 1). The sequence similarity value of the gyrB, rpoB, dnaK and recA housekeeping genes was also highest with M. lotononidis (93.6, 91.4, 88.2 and 83.4 %, respectively). These low sequence similarities of the four housekeeping genes also supported the hypothesis that strain NCCP-1258T belongs to a novel species.

Fig. 1
figure 1

Neighbor joining phylogenetic tree inferred from 16S rRNA gene sequences (1304 bp) showing inter-relationship of strain NCCP-1258T with the members of genus Microvirga and other closely related genera. Bootstrap values (>60 %) expressed as percentages of 1000 replications, are shown at the branch points. Solid circles represents nodes, which were recovered by three algorithms (NJ, MP and ML), whereas empty circles represents nodes, which were recovered by any of two algorithms. Azorhizobium caulinodans ORS 571T (AP009384) was rooted as outgroup. The length of the bar represents 1 % nucleotide sequence divergence

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Since the 16S rRNA gene sequence similarity of strain NCCP-1258T is higher than 97 % with four closely related type strains DDH was carried out as suggested by Stackebrandt and Goebel (1994). The results revealed that DDH values of strain NCCP-1258T were 41.6 % with the type strain Microvirga lotononidis WSM3557T, 39.2 % with M. lupini Lut6T, 33.6 % with M. zambiensis WSM3693T, and 15.4 % with M. flocculans ATCC BAA-817T. These values are less than the 70 % threshold that is indicative of the presence of new species (Wayne et al. 1987). The DNA G+C content of strain NCCP-1258T was determined to be 64.3 mol%, which is within the range of the members of genus Microvirga (Weon et al. 2010; Ardley et al. 2012).

Chemotaxonomic analyses

The cellular fatty acid profile of strain NCCP-1258T comprised predominantly of C18:1 ω7c (54.0 %), C19:0 cyclo ω8c (21.4 %), and C16:0 (14.5 %), was similar to the profiles obtained for the reference strains, although significant variation in the values of these components clearly differentiates our strain from the closely related reference strains (Table 2). It was noted that C17:0 cyclo and C18:1 ω7c were present in higher amounts, while C18:1 ω9c is absent in the profile of strain NCCP-1258T. Strain NCCP-1258T contained Q-10 as a sole menaquinone system. The closely related type strains were also reported to have Q-10 as the major menaquinone, Q-8 and Q-9 were present as minor components in these closely related reference strains but absent in strain NCCP-1258T. The polar lipids profile comprised of diphosphatidyl glycerol (DPG), phosphatidyl choline (PC), phosphatidyl dimethyl ethanolamine (PDE) and phosphatidyl ethanolamine (PE) (Supplementary Figure 3). Polar lipid profiles of the closely related strains M. lotononidis WSM3557T, M. lupini Lut6T, and M. zambiensis WSM3693T were also reported to contain these four components (Ardley et al. 2012). However, phosphatidyl glycerol (PG) is absent in strain NCCP-1258T which differentiated this isolate from the closely related taxa. On the basis of physiological, chemotaxonomic, phylogenetic, and genomic data, strain NCCP-1258T is considered to be a new member of the genus Microvirga.

Table 2 Cellular fatty acid profile (%) of strain NCCP-1258T in comparison with closely related type strains of the genus Microvirga
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Description of Microvirga pakistanensis sp. nov

Microvirga pakistanensis (pa.kis.tan.en’sis. N.L. fem. adj. pakistanensis is pertaining to Pakistan, where the type strain was isolated).

Cells are Gram-negative, aerobic, non-motile, and non-spore-forming short rods (~2 µm in length). Colonies are small, round with entire margins, shiny surface, concave, and pink in color. The optimum temperature, pH, and NaCl concentration for growth are 40 °C, 7.5–8.5 and 0.5 % (w/v), respectively. Positive for tryptophan deamination, nitrate reduction, oxidase, and hydrolysis of esculin, urea, and gelatine (weak), and fermentation of glucose but negative for Voges–Proskauer test. Acid was produced from arbutin, d-melibiose, salicin (weak), but not from d-cellobiose. Positive for assimilation of d-mannose, d-arabinose but negative for assimilation of l-arabinose and d-mannitol. Strong enzyme activity for esterase (C4), esterase lipase (C8) and lipase (C14) but negative for leucine arylamidase, α-chymotrypsin and napthol-As-BI-phosphohydrolase. d-fructose, l-fucose, and l-glutamic acid were utilized as carbon sources but negative for utilization of acetic acid. The major cellular fatty acids are C16:0, C18:1 ω7c, C19:0 cyclo ω8c, and summed feature 2 (C14:0 2-OH/iso-C16:1 I). The predominant menaquinone system is Q-10. The polar lipid profile comprises of diphosphatidylglycerol (DPG), phosphatidylcholine (PC), phosphatidyl dimethyl ethanolamine (PDE), and phosphatidyl ethanolamine (PE). The DNA G+C content is 64.3 mol%.

The type strain NCCP-1258T (=CGMCC 1.15074T = KCTC 42496T) was isolated from desert soil of Cholistan, Bahawalpur, Pakistan. GenBank accession numbers for type strain NCCP-1258T are LC065285 (16S rRNA gene); LC085517 (rpoB gene), LC085516 (gyrB gene), LC085515 (recA gene) and LC085514 (dnaK gene).