一株抗小鼠乳腺癌和一株有抑菌活性的北极细菌的初步研究

从采自北级的海泥、海水样品中共分离得到101株低温细菌,采用小鼠温敏型乳腺癌tsFT210细胞株和纸片扩散法对其进行了抗肿瘤和抑菌活性筛选,得到抗肿瘤活性细菌1株,抑茼活性菌株8株,并对其中抑菌活性较强的一株细菌AR084的适宜发酵条件、活性物质的稳定性进行了初步研究,确定了其培养条件。报道了北极海洋微生物的抗肿瘤和抑菌活性,且抑菌活性尚未见有报道。由此表明,极地微生物是潜在的活性物质的来源,在基础研究和开发应用方面具有广阔的前景。     

Genome and proteome characterization of the psychrophilic <Emphasis Type="Italic">Flavobacterium</Emphasis> bacteriophage 11b

Virion DNA of bacteriophage 11b (Φ11b), which infects a psychrophilic Flavobacterium isolate from Arctic sea-ice, was determined to consist of 36,012 bp. With 30.6% its GC content corresponds to that of host-genus species and is the lowest of all phages of Gram-negative bacteria sequenced so far. Similarities of several of 65 predicted ORFs, genome organization and phylogeny suggest an affiliation to ‘mesophilic’ nonmarine siphoviruses, e.g. to bacteriophages SPP1 and HK97. Early genes presumably encode an essential recombination factor (ERF), a single strand binding (SSB) protein, an endonuclease, and a DNA methylase. The late gene segment is likely to contain a terminase, portal, minor head, protease and a major capsid gene. Five ORFs exhibited similarities to Bacteroidetes species and seem to reflect the host specificity of the phage. Among PAGE-separated virion proteins that were identified by MALDI-ToF mass spectrometry are the portal, the major capsid, and a putative conserved tail protein. The Φ11b genome is the first to be described of a cultivated virus infecting a psychrophilic host as well as a Bacteroidetes bacterium. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Intrinsic halotolerance of the psychrophilic α-amylase from <Emphasis Type="Italic">Pseudoalteromonas haloplanktis</Emphasis>

The halotolerance of a cold adapted α-amylase from the psychrophilic bacterium Pseudoalteromonas haloplanktis (AHA) was investigated. AHA exhibited hydrolytic activity over a broad range of NaCl concentrations (0.01–4.5 M). AHA showed 28% increased activity in 0.5–2.0 M NaCl compared to that in 0.01 M NaCl. In contrast, the corresponding mesophilic (Bacillus amyloliquefaciens) and thermostable (B. licheniformis) α-amylases showed a 39 and 46% decrease in activity respectively. Even at 4.5 M NaCl, 80% of the initial activity was detected for AHA, whereas the mesophilic and thermostable enzymes were inactive. Besides an unaltered fluorescence emission and secondary structure, a 10°C positive shift in the temperature optimum, a stabilization factor of >5 for thermal inactivation and a ΔT _m of 8.3°C for the secondary structure melting were estimated in 2.7 M NaCl. The higher activation energy, half-life time and T _m indicated reduced conformational dynamics and increased rigidity in the presence of higher NaCl concentrations. A comparison with the sequences of other halophilic α-amylases revealed that AHA also contains higher proportion of small hydrophobic residues and acidic residues resulting in a higher negative surface potential. Thus, with some compromise in cold activity, psychrophilic adaptation has also manifested halotolerance to AHA that is comparable to the halophilic enzymes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This article is dedicated to Late Dr. P. V. Sundaram.     

Protein psychrophilicity: role of residual structural properties in adaptation of proteins to low temperatures

In order to investigate the structural distribution responsible for protein psychrophilicity, a systematic comparative analysis of 13 pairs of psychrophilic and mesophilic proteins is reported. Three kinds of residue structural states such as exposed, intermediate and buried were considered for analyzing the structural patterns of single amino acids and amino acids in different groups. The statistical test revealed that higher frequency in exposed state of Ala, higher frequency in intermediate state of His, lower frequency in buried state of Lys, lower frequency in exposed state of Gln, higher frequency in exposed state and in intermediate state of Thr, higher frequency in exposed and intermediate state of tiny and small amino acids groups could be critical factors related with protein psychrophilicity. Such structure-based differences of residual properties would help to develop a strategy for designing psychrophilic proteins.     

The stability of the TIM-barrel domain of a psychrophilic chitinase

Chitinase 60 from the psychrophilic bacterium Moritella marina (MmChi60) is a four-domain protein whose structure revealed flexible hinge regions between the domains, yielding conformations in solution that range from fully extended to compact. The catalytic domain is a shallow-grooved TIM-barrel. Heat-induced denaturation experiments of the wild-type and mutants resulting from the deletions of the two-Ig-like domains and the chitin binding domain reveal calorimetric profiles that are consistent with non-collaborative thermal unfolding of the individual domains, a property that must be associated to the “hinge-regions”. The calorimetric measurements of the (β/α)₈ catalytic domain reveal that the thermal unfolding is a slow-relaxation transition exhibiting a stable, partially structured intermediate state. Circular dichroism provides evidence that the intermediate exhibits features of a molten globule i.e., loss of tertiary structure while maintaining the secondary structural elements of the native. GdnHCl-induced denaturation studies of the TIM-barrel demonstrate an extraordinarily high resistance to the denaturant. Slow-relaxation kinetics characterize the unfolding with equilibration times exceeding six days, a property that is for the first time observed for a psychrophilic TIM barrel. On the other hand, the thermodynamic stability is ΔG=6.75±1.3 kcal/mol, considerably lower than for structural-insertions-containing barrels. The mutant E153Q used for the crystallographic studies of MmChi60 complexes with NAG ligands has a much lower stability than the wild-type.     

Chitinolytic bacteria and chitin mineralization in the marine waters and sediments along the Antartic Peninsula

Abstract Chitinolytic bacteria were enumerated and isolated from marine waters and sediments along the highly productive Antarctic Peninsula. Chitinolytic bacteria were found in low concentrations (approximately 1 cell per ml) in the water column and at much higher levels in marine surface sediments (10⁴–10⁵ per g). The predominant chitinolytic bacteria isolated from the water column were identified as psychrophilic Vibrio spp. Rates of chitin mineralization were measured by collection of ¹⁴CO₂ respired from ¹⁴C-labeled chitin synthesized from chitosan and [1-¹⁴C]acetic anhydride. Chitin mineralization rates were extremely low in the marine waters analyzed (0.00085–0.0019% of the added label respired in 48 h) and appreciably higher in the marine sediments (0.0039–0.01% per 48 h), suggesting that the sediments are much more important in chitin degradation. Such low mineralization rates suggest that chitin may be accumulating in Antarctic marine sediments, though animals may also play an important role in chitin degradation.     

Sequence analysis of a cryptic plasmid from Flavobacterium sp. KP1, a psychrophilic bacterium

A cryptic plasmid found at high copy number was isolated from Flavobacterium sp. KP1, a psychrophilic Gram-negative bacterium, cloned, and sequenced. The sequence will appear in the DDBJ/EMBL/GenBank databases under the accession number AB007196. The pFL1 plasmid is 2311 nucleotides in length with 32.7% GC content, and shows a distinctive nucleotide sequence without homology to other plasmids of similar length. The plasmid contains two open reading frames of significant length, ORFI and ORFII. ORFI encodes a protein similar to the replication proteins found in Gram-negative bacterial plasmids, Bacteroides fragilis plasmid pBI143 and Zymomonas mobilis plasmid pZM2. The putative translation product of ORFII shows homologies with plasmid recombination proteins found mainly in Gram-positive bacterial plasmids such as Staphylococcus aureus plasmid pT181.     

NH4 + transport system of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1

NH₄ ⁺ transport system of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 (Vibrio ABE-1) was examined by measuring the uptake of [¹⁴C]methylammonium ion (¹⁴CH₃NH^{3 +}) into the intact cells. ¹⁴CH₃NH₃ ⁺ uptake was detected in cells grown in medium containing glutamate as the sole nitrogen source, but not in those grown in medium containing NH₄Cl instead of glutamate. Vibrio ABE-1 did not utilize CH₃NH₃ ⁺ as a carbon or nitrogen source. NH₄Cl and nonradiolabeled CH₃NH₃ ⁺ completely inhibited ¹⁴CH₃NH₃ ⁺ uptake. These results indicate that ¹⁴CH₃NH₃ ⁺ uptake in this bacterium is mediated via an NH₄ ⁺ transport system and not by a specific carrier for CH₃NH₃ ⁺. The respiratory substrate succinate was required to drive ¹⁴CH₃NH₃ ⁺ uptake and the uptake was completely inhibited by KCN, indicating that the uptake was energy dependent. The electrochemical potentials of H⁺ and/or Na⁺ across membranes were suggested to be the driving forces for the transport system because the ionophores carbonylcyanide m-chlorophenylhydrazone and monensin strongly inhibited uptake activities at pH 6.5 and 8.5, respectively. Furthermore, KCl activated ¹⁴CH₃NH₃ ⁺ uptake. The ¹⁴CH₃NH₃ ⁺ uptake activity of Vibrio ABE-1 was markedly high at temperatures between 0° and 15°C, and the apparent K _m value for CH₃NH₃ ⁺ of the uptake did not change significantly over the temperature range from 0° to 25°C. Thus, the NH₄ ⁺ transport system of this bacterium was highly active at low temperatures. Received: August 1, 1998 / Accepted: October 8, 1998