Abstract
A comprehensive polyphasic taxonomic strategy was applied to the systematic characterization of strain DNPG3
T
, which was isolated from the river Ganges, Hooghly, West Bengal, India. The Gram-positive, halotolerant, heavy-metal-tolerant strain exhibited the ability to degrade
p
-nitrophenol (PNP). Cellular fatty acid analysis revealed that the predominant components were anteiso-C
15:0
(24.61%), C
11:0
(21.06%), iso-C
16:0
(11.89%), C
16:0
(11.58%), and anteiso-C
17:0
(11.24%). Notably, the presence of C
11:0
, C
10:0
2-OH as major fatty acids differentiate strain DNPG3
T
from its closely related members of the genus
Brachybacterium
. The predominant respiratory quinone was identified as menaquinone-7 (MK-7). Analysis of 16S rRNA gene sequence indicated that
B. zhongshanense
strain JB
T
was the closest relative of DNPG3
T
, sharing 97.08% sequence similarity. Genome-based ANI value calculated using the EzBioCloud server revealed that
B. zhongshanense
JCM 15471
T
was the closest genomic relative (85.49%). These values were further substantiated by digital DNA–DNA hybridization (dDDH) estimates calculated using the GGDC server. Taxonomic assignment using the GTDB database further indicated that strain DNPG3
T
constitutes a previously unrecognized species within the genus
Brachybacterium
. Genome analysis of strain DNPG3
T
identified eleven genomic islands, along with a rich repertoire of 194 carbohydrate-active enzyme (CAZyme) families, comprising 95 glycoside hydrolases and 53 glycosyltransferases. In addition, five biosynthetic gene clusters were detected. Collectively, these genomic features indicate the involvement of horizontal gene transfer events and highlighted the pronounced metabolic versatility of the strain, underscoring its potential for industrial enzyme production and secondary metabolite biosynthesis. Pan-genome analysis further indicates that the
Brachybacterium
pan-genome is open, reflecting substantial genetic diversity and ongoing gene acquisition within the genus. Comprehensive biochemical, physiological, chemotaxonomic, and phylogenetic analyses supported the assignment of strain DNPG3
T
to the genus
Brachybacterium
while clearly distinguishing it from all currently described species within the genus. Accordingly, strain DNPG3
T
was proposed to represent a novel species, for which the name
Brachybacterium netajii
sp. nov. is suggested. The type strain was DNPG3
T
(= MTCC13125
T
).