HLA-driven convergence of HIV-1 viral subtypes B and F toward the adaptation to immune responses in human populations

Background

Cytotoxic T-Lymphocyte (CTL) response drives the evolution of HIV-1 at a host-level by selecting HLA-restricted escape mutations. Dissecting the dynamics of these escape mutations at a population-level would help to understand how HLA-mediated selection drives the evolution of HIV-1.

Methodology/Principal Findings

We undertook a study of the dynamics of HIV-1 CTL-escape mutations by analyzing through statistical approaches and phylogenetic methods the viral gene gag sequenced in plasma samples collected between the years 1987 and 2006 from 302 drug-naïve HIV-positive patients. By applying logistic regression models and after performing correction for multiple test, we identified 22 potential CTL-escape mutations (p-value<0.05; q-value<0.2); 10 of these associations were confirmed in samples biologically independent by a Bayesian Markov Chain Monte-Carlo method. Analyzing their prevalence back in time we found that escape mutations that are the consensus residue in samples collected after 2003 have actually significantly increased in time in one of either B or F subtype until becoming the most frequent residue, while dominating the other viral subtype. Their estimated prevalence in the viral subtype they did not dominate was lower than 30% for the majority of samples collected at the end of the 80''s. In addition, when screening the entire viral region, we found that the 75% of positions significantly changing in time (p<0.05) were located within known CTL epitopes.

Conclusions

Across HIV Gag protein, the rise of polymorphisms from independent origin during the last twenty years of epidemic in our setting was related to an association with an HLA allele. The fact that these mutations accumulated in one of either B or F subtypes have also dominated the other subtype shows how this selection might be causing a convergence of viral subtypes to variants which are more likely to evade the immune response of the population where they circulate.     

First Phase I human clinical trial of a killed whole-HIV-1 vaccine: demonstration of its safety and enhancement of anti-HIV antibody responses

Background

Vaccination with inactivated (killed) whole-virus particles has been used to prevent a wide range of viral diseases. However, for an HIV vaccine this approach has been largely negated due to inherent safety concerns, despite the ability of killed whole-virus vaccines to generate a strong, predominantly antibody-mediated immune response in vivo. HIV-1 Clade B NL4-3 was genetically modified by deleting the nef and vpu genes and substituting the coding sequence for the Env signal peptide with that of honeybee melittin signal peptide to produce a less virulent and more replication efficient virus. This genetically modified virus (gmHIV-1_NL4-3) was inactivated and formulated as a killed whole-HIV vaccine, and then used for a Phase I human clinical trial (Trial Registration: Clinical Trials NCT01546818). The gmHIV-1_NL4-3 was propagated in the A3.01 human T cell line followed by virus purification and inactivation with aldrithiol-2 and γ-irradiation. Thirty-three HIV-1 positive volunteers receiving cART were recruited for this observer-blinded, placebo-controlled Phase I human clinical trial to assess the safety and immunogenicity.

Results

Genetically modified and killed whole-HIV-1 vaccine, SAV001, was well tolerated with no serious adverse events. HIV-1_NL4-3-specific PCR showed neither evidence of vaccine virus replication in the vaccine virus-infected human T lymphocytes in vitro nor in the participating volunteers receiving SAV001 vaccine. Furthermore, SAV001 with adjuvant significantly increased the pre-existing antibody response to HIV-1 proteins. Antibodies in the plasma of vaccinees were also found to recognize HIV-1 envelope protein on the surface of infected cells as well as showing an enhancement of broadly neutralizing antibodies inhibiting tier I and II of HIV-1 B, D, and A subtypes.

Conclusion

The killed whole-HIV vaccine, SAV001, is safe and triggers anti-HIV immune responses. It remains to be determined through an appropriate trial whether this immune response prevents HIV infection.

     

Single nucleotide polymorphisms in <Emphasis Type="Italic">TaER</Emphasis> genes and their association with carbon isotope discrimination in wheat genotypes under drought

Candidate gene association studies implicate the detection of contributing single nucleotide polymorphism (SNP) for the target traits and have been recommended as a promising technique to anatomize the complex characters in plants. The ERECTA gene in plants controls different physiological functions. In this study, we identified SNPs in 1.1 kb partial sequences of TaER-1 and TaER-2 of wheat (Triticum aestivum L.). Thirty-nine SNPs were identified in the coding regions of TaER-1 gene in 33 wheat genotypes, of which 20 SNPs caused non-synonymous mutations while 19 SNPs produced synonymous mutations; 31 SNPs were located in the coding regions of TaER-2 gene in 26 genotypes, of which 18 SNPs caused non-synonymous mutations and 13 SNPs caused synonymous mutations. In addition, 32 SNPs in TaER-1 and 9 SNPs in TaER-2 were also identified in the non-coding regions. Moreover, the significant genetic associations of SNPs of TaER-1 and TaER-2 genes with carbon isotope discrimination, stomatal conductance, photosynthetic rate, transpiration rate, intrinsic water use efficiency (iWUE), leaf length, leaf width, stomatal density, epidermal cell density, and stomatal index were noted in wheat genotypes. This study confirms the importance of TaER-1 and TaER-2 genes which could improve iWUE of wheat by regulating leaf gas exchange and leaf structural traits. These identified SNPs may play a critical role in molecular breeding by means of marker-assisted selection.     

Mesophyll conductance to CO<Subscript>2</Subscript> in leaves of Siebold’s beech (<Emphasis Type="Italic">Fagus crenata</Emphasis>) seedlings under elevated ozone

Ozone is an air pollutant that negatively affects photosynthesis in woody plants. Previous studies suggested that ozone-induced reduction in photosynthetic rates is mainly attributable to a decrease of maximum carboxylation rate (V_cmax) and/or maximum electron transport rate (J_max) estimated from response of net photosynthetic rate (A) to intercellular CO₂ concentration (C_i) (A/C_i curve) assuming that mesophyll conductance for CO₂ diffusion (g_m) is infinite. Although it is known that C_i-based V_cmax and J_max are potentially influenced by g_m, its contribution to ozone responses in C_i-based V_cmax and J_max is still unclear. In the present study, therefore, we analysed photosynthetic processes including g_m in leaves of Siebold’s beech (Fagus crenata) seedlings grown under three levels of ozone (charcoal-filtered air or ozone at 1.0- or 1.5-times ambient concentration) for two growing seasons in 2016–2017. Leaf gas exchange and chlorophyll fluorescence were simultaneously measured in July and September of the second growing season. We determined the A, stomatal conductance to water vapor and g_m, and analysed A/C_i curve and A/C_c curve (C_c: chloroplast CO₂ concentration). We also determined the Rubisco and chlorophyll contents in leaves. In September, ozone significantly decreased C_i-based V_cmax. At the same time, ozone decreased g_m, whereas there was no significant effect of ozone on C_c-based V_cmax or the contents of Rubisco and chlorophyll in leaves. These results suggest that ozone-induced reduction in C_i-based V_cmax is a result of the decrease in g_m rather than in carboxylation capacity. The decrease in g_m by elevated ozone was offset by an increase in C_i, and C_c did not differ depending on ozone treatment. Since C_c-based V_cmax was also similar, A was not changed by elevated ozone. We conclude that g_m is an important factor for reduction in C_i-based V_cmax of Siebold’s beech under elevated ozone.     

Re-visiting the functional Relevance of the highly conserved Serine 40 Residue within HIV-1 p6<Superscript>Gag</Superscript>

Background

HIV-1 formation is driven by the viral structural polyprotein Gag, which assembles at the plasma membrane into a hexagonal lattice. The C-terminal p6^Gag domain harbors short peptide motifs, called late domains, which recruit the cellular endosomal sorting complex required for transport and promote HIV-1 abscission from the plasma membrane. Similar to late domain containing proteins of other viruses, HIV-1 p6 is phosphorylated at multiple residues, including a highly conserved serine at position 40. Previously published studies showed that an S40F exchange in p6^Gag severely affected virus infectivity, while we had reported that mutation of all phosphorylatable residues in p6^Gag had only minor effects.

Findings

We introduced mutations into p6^Gag without affecting the overlapping pol reading frame by using an HIV-1 derivative where gag and pol are genetically uncoupled. HIV-1 derivatives with a conservative S40N or a non-conservative S40F exchange were produced. The S40F substitution severely affected virus maturation and infectivity as reported before, while the S40N exchange caused no functional defects and the variant was fully infectious in T-cell lines and primary T-cells.

Conclusions

An HIV-1 variant carrying a conservative S40N exchange in p6^Gag is fully functional in tissue culture demonstrating that neither S40 nor its phosphorylation are required for HIV-1 release and maturation. The phenotype of the S40F mutation appears to be caused by the bulky hydrophobic residue introduced into a flexible region.

     

Polyclonal Antibodies in Microplates to Predict the Maximum Adsorption Activities of Enzyme/Mutants from Cell Lysates

With microplate-immobilized polyclonal antibodies against a starting enzyme or its active mutant bearing consistent accessible epitopes, the maximum activity of an adsorbed enzyme/mutant (Vs) was predicted for comparison to recognize weakly-positive mutants. Rabbit antisera against Escherichia coli alkaline phosphatase (ECAP) were fractionated with 33% ammonium sulfate to yield crude polyclonal antibodies for conventional immobilization in 96-well microplates. The response curve of the activities of ECAP/mutant adsorbed by the immobilized polyclonal antibodies to protein quantities from a cell lysate was fit to an approximation model to predict Vs. With 0.4 μg crude polyclonal antibody for immobilization, Vs was consistent for ECAP in cell lysates bearing fourfold differences in its apparent specific activities when its abundance was greater than 0.9%. The ratio of Vs of the mutant R168K to that of ECAP was 1.5?±?0.1 (n?=?2), consistent with that of their specific activities after affinity purification. Unfortunately, the prediction of Vs with polyclonal antibodies that saturated microplate wells was ineffective to Pseudomonas aeruginosa arylsulfatase bearing less than 2% specific activity of ECAP. Therefore, with microplate-immobilized polyclonal antibodies to adsorb enzyme/mutants from cell lysates, high-throughput prediction of Vs was practical to recognize weakly-positive mutants of starting enzymes bearing fairly-high activities.     

Evidences and magnitude of nighttime transpiration derived from <Emphasis Type="Italic">Populus euphratica</Emphasis> in the extreme arid region of China

Extensive research has found that nighttime transpiration (E _n) is positively correlated to the vapour pressure deficit (VPD), that suggested E _n was highest during the night under high temperatures and low humidity along with high soil water availability, typically for the riparian forest in the extreme arid region of China. This study used the heat ratio method to measure sap velocity (V _s) for mature and saplings Populus euphratica Oliv., and then E _n was conservatively calculated as total nocturnal sap flow (F _s, the product of V _s and sapwood area A _s) between 01:00 to 06:00. A gas exchange system was used to measure the leaf transpiration rate (T _r) and stomatal conductance (g _s) of saplings. For mature trees, nighttime V _s was extensive and logarithmic correlated to VPD (similar to daytime). For saplings, g _s and T _r was extensive in different months, and also a strong logarithmic relationship was found between V _s and VPD for both daytime and nighttime periods. Both of stem sap flow and leaf gas exchange suggusted the occurrence of E _n, whether mature or sapling trees. E _n contribution to daily transpiration (E _d) was high just as expected for P. euphratica, which was confirmed by proportional E _n to E _d (E _n/E _d) means taken in 2012 (24.99%) and 2013 (34.08%). Compared to mature trees, E _n/E _d of saplings in 2013 was lower with means of 12.06%, that supported further by the shorter duration times and less T _r,n (16.64%) and g _s,n (26.45%) of leaf, suggesting that E _n magnitude is associated to individual the tree size, that effect to stored water of individual trees, although this hypothesis requires further research.     

Next generation genetic mapping of the Ligon-lintless-2 (<Emphasis Type="Italic">Li</Emphasis><Subscript>2</Subscript>) locus in upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Key message

Mapping-by-sequencing and novel subgenome-specific SNP markers were used to fine map the Ligon-lintless 2 ( Li ₂ ) short-fiber gene in tetraploid cotton. These methodologies will accelerate gene identification in polyploid species.

Abstract

Next generation sequencing offers new ways to identify the genetic mechanisms that underlie mutant phenotypes. The release of a reference diploid Gossypium raimondii (D₅) genome and bioinformatics tools to sort tetraploid reads into subgenomes has brought cotton genetic mapping into the genomics era. We used multiple high-throughput sequencing approaches to identify the relevant region of reference sequence and identify single nucleotide polymorphisms (SNPs) near the short-fiber mutant Ligon-lintless 2 (Li ₂) gene locus. First, we performed RNAseq on 8-day post-anthesis (DPA) fiber cells from the Li ₂ mutant and its wild type near isogenic line (NIL) Gossypium hirsutum cv. DP5690. We aligned sequence reads to the D₅ genome, sorted the reads into A and D subgenomes with PolyCat and called SNPs with InterSNP. We then identified SNPs that would result in non-synonymous substitutions to amino acid sequences of annotated genes. This step allowed us to identify a 1-Mb region with 24 non-synonymous SNPs, representing the introgressed region that differentiates Li ₂ from its NIL. Next, we sequenced total DNA from pools of F₂ plants, using a super bulked segregant analysis sequencing (sBSAseq) approach. The sBSAseq predicted 82 non-synonymous SNPs among 3,494 SNPs in a 3-Mb region that includes the region identified by RNAseq. We designed subgenome-specific SNP markers and tested them in an F₂ population of 1,733 individuals to construct a genetic map. Our resulting genetic interval contains only one gene, an aquaporin, which is highly expressed in wild-type fibers and is significantly under-expressed in elongating Li ₂ fiber cells.

     

The use of parameters of chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence induction to evaluate the state of plants under anthropogenic load

The physiological state of the leaves of the small-leaved linden (Tilia cordata), silver birch (Betula pendula), and northern white cedar (Thuja occidentalis) under urban conditions was assessed via recording the kinetics of chlorophyll under fluorescence induction. Different sensitivities of the plants to adverse growing conditions were revealed. The most sensitive parameters of the fluorescence JIP test, viz., PI _ABS , F _V/F ₀, and F _V/F _M, were identified as indicators of the physiological state of the urban phytocoenosis. Recommendations for the application of the method for monitoring studies are presented.     

Volatile compounds from beneficial or pathogenic bacteria differentially regulate root exudation,transcription of iron transporters,and defense signaling pathways in <Emphasis Type="Italic">Sorghum bicolor</Emphasis>

Key message

Our results show that Sorghum bicolor is able to recognize bacteria through its volatile compounds and differentially respond to beneficial or pathogens via eliciting nutritional or defense adaptive traits.

Abstract

Plants establish beneficial, harmful, or neutral relationships with bacteria. Plant growth promoting rhizobacteria (PGPR) emit volatile compounds (VCs), which may act as molecular cues influencing plant development, nutrition, and/or defense. In this study, we compared the effects of VCs produced by bacteria with different lifestyles, including Arthrobacter agilis UMCV2, Bacillus methylotrophicus M4-96, Sinorhizobium meliloti 1021, the plant pathogen Pseudomonas aeruginosa PAO1, and the commensal rhizobacterium Bacillus sp. L2-64, on S. bicolor. We show that VCs from all tested bacteria, except Bacillus sp. L2-64, increased biomass and chlorophyll content, and improved root architecture, but notheworthy A. agilis induced the release of attractant molecules, whereas P. aeruginosa activated the exudation of growth inhibitory compounds by roots. An analysis of the expression of iron-transporters SbIRT1, SbIRT2, SbYS1, and SbYS2 and genes related to plant defense pathways COI1 and PR-1 indicated that beneficial, pathogenic, and commensal bacteria could up-regulate iron transporters, whereas only beneficial and pathogenic species could induce a defense response. These results show how S. bicolor could recognize bacteria through their volatiles profiles and highlight that PGPR or pathogens can elicit nutritional or defensive traits in plants.

     

Longitudinal assessment of the effect of concentration on stream N uptake rates in an urbanizing watershed

We examined the effect of concentration on nitrogen uptake patterns for a suburban stream in Maryland and addressed the question: How does NO₃ ^? uptake change as a function of concentration and how do uptake patterns compare with those found for NH₄ ⁺? We applied a longitudinal (stream channel corridor) approach in a forested stream section and conducted short-term nutrient addition experiments in late summer 2004. In the downstream direction, NO₃ ^? concentrations decreased because of residential development in headwaters and downstream dilution; NH₄ ⁺ concentrations slightly increased. The uptake patterns for NO₃ ^? were very different from NH₄ ⁺. While NH₄ ⁺ had a typical negative relationship between first-order uptake rate constant (K _c ) and stream size, NO₃ ^? had a reverse pattern. We found differences for other metrics, including uptake velocity (V _f ) and areal uptake rate (U). We attributed these differences to a stream size effect, a concentration effect and a biological uptake capacity effect. For NO₃ ^? these combined effects produced a downstream increase in K _c , V _f and U; for NH₄ ⁺ they produced a downstream decrease in K _c and V _f , and a not well defined pattern for U. We attributed a downstream increase in NO₃ ^? uptake capacity to an increase in hyporheic exchange and a likely increase in carbon availability. We also found that K _c and V _f were indirectly related with concentration. Similar evidence of ‘nutrient saturation’ has been reported in other recent studies. Our results suggest that higher-order uptake models might be warranted when scaling NO₃ ^? uptake across watersheds that are subject to increased nitrogen loading.     

Subcutaneous administration CpG-ODNs acts as a potent adjuvant for an HIV-1-<Emphasis Type="Italic">tat</Emphasis>-based vaccine candidate to elicit cellular immunity in BALB/c mice

Objective

To evaluate the combined effects of CpG oligodeoxynucleotides (CpG-ODNs) adjuvant and subcutaneous injection route on efficacy of a HIV-1-tat DNA vaccine candidate using BALB/c mice as an animal model.

Results

Evaluation of cellular and humoral immunity of mice injected subcutaneously with HIV-1-tat gene cloned into a pcDNA3.1 vector indicated that significant levels of IFN-γ cytokine secretion (900 pg/ml), lymphocyte proliferation (2.5 stimulation index) and IgG_2a (1.45 absorbance 450 nm) production could be achieved. These indicators of stimulated cellular immunity were elicited 2 weeks after the last injection (P < 0.05).

Conclusions

Formulation of HIV-1-tat DNA vaccine candidate with CpG-ODNs as an adjuvant while administrated subcutaneously are a promising approach to induce effective cellular immunity responses against HIV-1 infection.

     

Calponin-like protein from mussel smooth muscle is a competitive inhibitor of actomyosin ATPase

The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters V_max and K_ATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in K_ATPase value and to a lesser extent the decrease in V_max, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of V_max and K_ATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.     

Altered somatic mutation level and DNA repair gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> exposed to ultraviolet C,salt, and cadmium stresses

It is known that somatic mutations arising during animal growth and ageing contribute to the development of neurodegenerative and other animal diseases. For plants, several studies showed that small-scale somatic DNA mutations accumulated during Arabidopsis life cycle. However, there is a lack of data on the influence of environmental stresses on somatic DNA mutagenesis in plants. In this study, we analyzed the effects of ultraviolet C (UV-C) irradiation, high soil salinity, and cadmium (CdI₃) stresses on the level of small-scale somatic DNA mutations in Arabidopsis thaliana. The number of DNA mutations was examined in the Actin2 3′UTR (Actin-U1), ITS1-5.8rRNA-ITS2 (ITS), and ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) DNA regions. We found that somatic mutation levels considerably increased in CdI₃-treated Arabidopsis plants, while the mutation levels declined in the UV-C- and NaCl-treated A. thaliana. Cadmium is a mutagen that is known to inhibit DNA repair processes. The detected stress-induced alterations in somatic DNA mutation levels were accompanied by markedly increased expression of base excision repair genes (AtARP, AtDME, AtDML2, AtDML3, AtMBD4, AtROS, AtUNG, and AtZDP), nucleotide excision repair genes (AtDDB1a, AtRad4, and AtRad23a), mismatch repair genes (AtMSH2, AtMSH3, and AtMSH7), and photoreactivation genes (AtUVR2, AtUVR3). Thus, the results demonstrated that UV-C, high soil salinity, and cadmium stresses influence both the level of DNA mutations and expression of DNA repair genes. Salt- and UV-induced activation of DNA repair genes could contribute to the stress-induced decrease in somatic mutation level.     

Catalytic properties of aminoacylase of strain <Emphasis Type="Italic">Rhodococcus armeniensis</Emphasis> AM6.1

Studies of substrate specificity revealed that the D-aminoacylase of Rhodococcus armeniensis AM6.1 strain exhibits absolute stereospecificity to the D-stereoisomers of N-acetyl-amino acids. The enzyme is the most active reacted with N-acetyl-D-methionine, as well as with aromatic and hydrophobic N-acetylamino acids and interacts weakly with the basic substrates. It is practically not reacted with acidic and hydrophilic N-acetyl-amino acids. Michaelis constants (K_m) and maximum reaction velocities (V_max) were calculated, using linear regression analysis, for the following substrates: N-acetyl-D-methionine, N-acetyl-D-alanine, N-acetyl-D-phenylalanine, N-acetyl-D-tyrosine, N-acetyl-D-valine, N-acetyl-D-oxyvaline, N-acetyl- D-leucine. Substrate inhibition of D-aminoacylase was displayed with N-acetyl-D-leucine (K_s = 35.5 ± 28.3 mM) and N-acetyl-DL-tyrosine (K_s = 15.8 ± 4.5 mM). Competitive inhibition of the enzyme with product–acetic acid (K_i = 104.7 ± 21.7 mM, K_m = 2.5 ± 0.5 mM, V_max = 25.1 ± 1.5 U/mg) was observed.     

Inheritance of Traits Controlled by Odd Chromosomes Using Data on Transmission of Monosomic Addition S<Superscript>t</Superscript> Chromosome of the <Emphasis Type="Italic">Elymus Sibiricus</Emphasis> Genome

On the basis of the winter bread wheat cultivar Obryi, two independent disomic addition lines BC₁₂F_∞ with the chromosome of the E. sibiricus S^t genome are created. A practical algorithm for determining the probabilities of transmission of the odd chromosome separately through male and female gametes in selfpollination of hemizygous hybrids from the equation p²–(1 + f₁–f₄) × p + f₁ = 0 is proposed, where p is the probability of the formation of viable gametes with the considered chromosome and f₁ and f₄ are the empirical frequencies of the corresponding homozygotes with and without the trait. The probability of transmission of an alien univalent chromosome through pollen (p_♂) is associated with the frequency of its transmission through the egg cell (p_♀) in backcrosses and in self-pollination (1–f₄) by the equation p_♂ = 1–f₄/(1–p_♀). The calculated empirically dependent estimates of the probabilities of transmission of the added chromosome through the egg cell p_♀ = 18.7% and through pollen p_♂ = 4.3% correspond to the empirical frequencies obtained for backcrosses. The coefficients of the gamete selection V_♀ = 0.748 and V_♂ = 0.172 are calculated, and the expected segregation for the alien trait controlled by a dominant gene located in the added chromosome is determined—with the trait: without the trait is 0.222: 0.778 in F₂; 0.187: 0.813 in equational and 0.043: 0.957 in certational backcrosses.     

Genetic characterization of phenicol-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> and role of wild-type repressor/regulator gene (<Emphasis Type="Italic">acrR</Emphasis>) on phenicol resistance

The genetic basis for phenicol resistance was examined in 38 phenicol-resistant clinical Escherichia coli isolates from poultry. Out of 62 isolates, 38 showed resistance for chloramphenicol and nine for florfenicol, respectively. Each strain also demonstrated resistance to a variety of other antibiotics. Molecular detection revealed that the incidence rates of the cat1, cat2, flo, flo-R, cmlA, and cmlB were 32, 29, 18, 13, 0, and 0%, respectively. Nineteen strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of five isolates revealed the amino acid changes in four isolates. DNA sequencing showed the non-synonymous mutations which change the amino acid, silent mutation, and nucleotide deletion in four isolates. MY09C10 showed neither deletion nor mutation in nucleotide. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in these strains. Complementation with a plasmid-borne wild-type acrR gene reduced the expression level of AcrA protein in the mutants and partially restored antibiotic susceptibility one- to fourfold. This study shows that mutations in acrR are an additional genetic basis for phenicol resistance.