- A new high-throughput culturing (HTC) method using a low nutrient
heterotrophic medium (LNHM) has led to the isolation of many novel strains of
oligotrophic bacteria from marine ecosystems. Four strains belonging to a single
dade, HTCC2151, HTCC218OT, HTCC2178T and HTCC2188T, were isolated
from the coast of Oregon by the HTC method. Phylogenetic analysis based on
their 1 6S rDNA sequences showed that they fell into the OM1 82 dade in the
oligotrophic marine Gammaproteobacteria (0MG) group, which is distantly
related to previously cultivated genera in Gammaproteobacteria. This analysis,
along with DNA-DNA hybridization results, indicated that strains HTCC2151 and
HTCC218OT were the same species, showing 89.6% genomic DNA relatedness
between these strains. Strain HTCC2178T was revealed to fall into the same genus
with strains HTCC2 151 and HTCC2 180T, but a different species, and this was
supported by 96.5% 96.7% of 16S rRNA gene sequence similarity with the
strains and 27.4% of genomic DNA relatedness between strains HTCC2 178T and
HTCC218OT. Strain HTCC2188T differed significantly from HTCC2151,
HTCC218OT and HTCC2178T, with less than 12.1% of the DNA-DNA
hybridization result with any of the strains, and thus it was proposed that strain
HTCC2188T be placed in a separate genus in the 0M182 dade. G+C mol% of strains HTCC2151, HTCC218OT, HTCC2178T and HTCC2188T were 36.2%, 41%,
54.2% and 49.3%, respectively (HPLC method).
All the HTCC strains in 0M182 dade were Gram-negative, and
microscopic observations revealed that they were short rods of 0.06 xm3 to 0.13
m3 in size on average and divided by binary fission. Growth curves and specific
growth rates at six different temperatures and six different mixed carbon
concentrations were determined. Their growth characteristics showed that these
bacteria were slowly growing, obligate or facultative oligotrophic and psychro-tomesophilic
Two of the strains, HTCC2178T and HTCC2188T, started forming tiny
colonies on l/1OR2A at 16°C after a 10-day incubation, but not HTCC2151 and
HTCC2 180T Five different liquid artificial seawater (A SW) media were tested for
their ability to support growth of the strains, and strains HTCC218OT, HTCC2 178T
and HTCC2188T grew in one or two different ASW media, but not HTCC215O.
pH ranges for growth of strains HTCC2151, HTCC218OT, HTCC2178T and
HTCC2188T were pH 7-8.5, pH 7.5-10, pH 7-11, andpll 7.5 10. Ranges of
NaCI concentrations for growth of strains HTCC2 180T, HTCC2 178T and
HTCC2188Twere 1.5-3.3%, 1.0-3.3%, and2.0-3.3%.
Thirty four different carbon sources were utilized by strain HTCC2 180T,
fourteen by HTCC2178T and fifteen by strain HTCC21S8T as sole carbon source
among forty seven different carbon sources. Strain HTCC2180T utilized all kinds
of hexoses and sugar alcohols tested, most of pentoses, oligosaccharides and
organic acids, some of amino acids, and one of amino sugars as sole carbon
sources. Most of pentoses and hexoses, some of oligosaccharides and organic
acids, and only one kind of sugar alcohols and amino acids were utilized by strain HTCC2178T, however, none of amino sugars. Strain HTCC2188T utilized most of
hexoses, some of pentoses, oligosaccharides and sugar alcohols, and one of amino
acids and amino sugars, but none of organic acids was utilized by this strain as
sole carbon source.
All the strains in the OM 182 dade showed positive reactions for alkaline
phosphatase, esterase, esterase lipase and naphthol-AS-BI-phosphohydrolase.
Presence of acid phosphatase and naphthol-AS-BI-phosphohydrolase was detected
from strains HTCC2151 and HTCC218OT. Only one strain, HTCC2188T,
exhibited positive results for leucine arylamidase, a-chymotrypsin and N-acetyl-f3-
HTCC strains in the 0M182 dade contained the fatty acids, 16:0, 18:0 and
16:1, commonly found in previously cultivated genera in Gammaproteobacteria.
They also showed the presence of 17:1 o7c or 17:1 w8c as one of the major fatty
acids, which was not usually found as a dominant fatty acid in other genera in
From the genotypic and polyphasic evidences, it is proposed that strains
HTCC2151, HTCC218OT and HTCC2178T should be placed into a new genus
named Marinivirgula gen. nov. including Marinivirgulafluito gen. nov., sp. nov.
with the type strain HTCC2 180T, and Marinivirgula obesa gen. nov., sp. nov. with
the type strain HTCC2178T. The name Litincolaparvulus gen. nov., sp. nov. is
suggested for the type strain HTCC2 188 T.